Modulators of amyloid aggregation

ABSTRACT

Compounds that modulate the aggregation of amyloidogenic proteins or peptides are disclosed. The modulators of the invention can promote amyloid aggregation or, more preferably, can inhibit natural amyloid aggregation. In a preferred embodiment, the compounds modulate the aggregation of natural β amyloid peptides (β-AP). In a preferred embodiment, the β amyloid modulator compounds of the invention are comprised of an Aβ aggregation core domain and a modifying group coupled thereto such that the compound alters the aggregation or inhibits the neurotoxicity of natural β amyloid peptides when contacted with the peptides. Furthermore, the modulators are capable of altering natural β-AP aggregation when the natural β-APs are in a molar excess amount relative to the modulators. Pharmaceutical compositions comprising the compounds of the invention, and diagnostic and treatment methods for amyloidogenic diseases using the compounds of the invention, are also disclosed.

RELATED APPLICATIONS

[0001] This application is a continuation of patent application Ser. No.09/972,475, filed on Oct. 4, 2001, which is a continuation of patentapplication Ser. No. 08/617,267, filed on Mar. 14, 1996, which is acontinuation-in-part of U.S. patent application Ser. No. 08/404,831,filed Mar. 14, 1995, and U.S. patent application Ser. No. 08/475,579,filed Jun. 7, 1995 and U.S. patent application Ser. No. 08/548,988,filed Oct. 27, 1995, the entire contents of each of which are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

[0002] Alzheimer's disease (AD), first described by the Bavarianpsychiatrist Alois Alzheimer in 1907, is a progressive neurologicaldisorder that begins with short term memory loss and proceeds todisorientation, impairment of judgement and reasoning and, ultimately,dementia. The course of the disease usually leads to death in a severelydebilitated, immobile state between four and 12 years after onset. ADhas been estimated to afflict 5 to 11 percent of the population over age65 and as much as 47 percent of the population over age 85. The societalcost for managing AD is upwards of 80 billion dollars annually,primarily due to the extensive custodial care required for AD patients.Moreover, as adults born during the population boom of the 1940's and1950's approach the age when AD becomes more prevalent, the control andtreatment of AD will become an even more significant health careproblem. Currently, there is no treatment that significantly retards theprogression of the disease. For reviews on AD, see Selkoe, D. J. Sci.Amer., November 1991, pp. 68-78; and Yankner, B. A. et al. (1991) N.Eng. J. Med. 325:1849-1857.

[0003] It has recently been reported (Games et al. (1995) Nature373:523-527) that an Alzheimer-type neuropathology has been created intransgenic mice. The transgenic mice express high levels of human mutantamyloid precursor protein and progressively develop many of thepathological conditions associated with AD.

[0004] Pathologically, AD is characterized by the presence ofdistinctive lesions in the victim's brain. These brain lesions includeabnormal intracellular filaments called neurofibrillary tangles (NTFs)and extracellular deposits of amyloidogenic proteins in senile, oramyloid, plaques. Amyloid deposits are also present in the walls ofcerebral blood vessels of AD patients. The major protein constituent ofamyloid plaques has been identified as a 4 kilodalton peptide calledβ-amyloid peptide (β-AP) (Glenner, G. G. and Wong, C. W. (1984) Biochem.Biophys. Res. Commun. 120:885-890; Masters, C. et al. (1985) Proc. Natl.Acad. Sci. USA 82:4245-4249). Diffuse deposits of β-AP are frequentlyobserved in normal adult brains, whereas AD brain tissue ischaracterized by more compacted, dense-core β-amyloid plaques. (Seee.g., Davies, L. et al. (1988) Neurology 38:1688-1693) Theseobservations occurs in AD. In further support of a direct pathogenicrole for β-AP, β-amyloid has been shown to be toxic to mature neurons,both in culture and in vivo. Yankner, B. A. et al. (1989) Science245:417-420; Yankner, B. A. et al. (1990) Proc. Natl. Acad. Sci. USA87:9020-9023; Roher, A. E. et al. (1991) Biochem. Biophys. Res. Commun.174:572-579; Kowall, N. W. et al. (1991) Proc. Natl. Acad. Sci. USA88:7247-7251. Furthermore, patients with hereditary cerebral hemorrhagewith amyloidosis-Dutch-type (HCHWA-D), which is characterized by diffuseβ-amyloid deposits within the cerebral cortex and cerebrovasculature,have been shown to have a point mutation that leads to an amino acidsubstitution within β-AP. Levy, E. et al. (1990) Science 248:1124-1126.This observation demonstrates that a specific alteration of the β-APsequence can cause β-amyloid to be deposited.

[0005] Natural β-AP is derived by proteolysis from a much larger proteincalled the amyloid precursor protein (APP). Kang, J. et al. (1987)Nature 325:733; Goldgaber, D. et al. (1987) Science 235:877; Robakis, N.K. et al. (1987) Proc. Natl. Acad. Sci. USA 84:4190; Tanzi, R. E. et al.(1987) Science 235:880. The APP gene maps to chromosome 21, therebyproviding an explanation for the β-amyloid deposition seen at an earlyage in individuals with Down's syndrome, which is caused by trisomy ofchromosome 21. Mann, D. M. et al. (1989) Neuropathol. Appl. Neurobiol.15:317; Rumble, B. et al. (1989) N. Eng. J. Med. 320:1446. APP containsa single membrane spanning domain, with a long amino terminal region(about two-thirds of the protein) extending into the extracellularenvironment and a shorter carboxy-terminal region projecting into thecytoplasm. Differential splicing of the APP messenger RNA leads to atleast five forms of APP, composed of either 563 amino acids (APP-563),695 amino acids (APP-695), 714 amino acids (APP-714), 751 amino acids(APP-751) or 770 amino acids (APP-770).

[0006] Within APP, naturally-occurring β amyloid peptide begins at anaspartic acid residue at amino acid position 672 of APP-770.Naturally-occurring β-AP derived from proteolysis of APP is 39 to 43amino acid residues in length, depending on the carboxy-terminal endpoint, which exhibits heterogeneity. The predominant circulating form ofβ-AP in the blood and cerebrospinal fluid of both AD patients and normaladults is β1-40 (“short β”). Seubert, P. et al. (1992) Nature 359:325;Shoji, M. et al. (1992) Science 258:126. However, β1-42 and β1-43 (“longβ”) also are forms in β-amyloid plaques. Masters, C. et al. (1985) Proc.Natl. Acad. Sci. USA 82:4245; Miller, D. et al. (1993) Arch. Biochem.Biophys. 301:41; Mori, H. et al. (1992) J. Biol. Chem. 267:17082.Although the precise molecular mechanism leading to β-APP aggregationand deposition is unknown, the process has been likened to that ofnucleation-dependent polymerizations, such as protein crystallization,microtubule formation and actin polymerization. See e.g., Jarrett, J. T.and Lansbury, P. T. (1993) Cell 73:1055-1058. In such processes,polymerization of monomer components does not occur until nucleusformation. Thus, these processes are characterized by a lag time beforeaggregation occurs, followed by rapid polymerization after nucleation.Nucleation can be accelerated by the addition of a “seed” or preformednucleus, which results in rapid polymerization. The long β forms of β-APhave been shown to act as seeds, thereby accelerating polymerization ofboth long and short β-AP forms. Jarrett, J. T. et al. (1993)Biochemistry 32:4693.

[0007] In one study, in which amino acid substitutions were made inβ-AP, two mutant β peptides were reported to interfere withpolymerization of non-mutated β-AP when the mutant and non-mutant formsof peptide were mixed. Hilbich, C. et al. (1992) J. Mol. Biol.228:460-473. However, equimolar amounts of the mutant and non-mutant(i.e., natural), amyloid peptides were used to see this effect and themutant peptides were reported to be unsuitable for use in vivo. Hilbich,C. et al. (1992), supra.

SUMMARY OF THE INVENTION

[0008] This invention pertains to compounds, and pharmaceuticalcompositions thereof, that can modulate the aggregation of amyloidogenicproteins and peptides, in particular compounds that can modulate theaggregation of natural β amyloid peptides (β-AP) and inhibit theneurotoxicity of natural β-APs. In one embodiment, the inventionprovides an amyloid modulator compound comprising an amyloidogenicprotein, or peptide fragment thereof, coupled directly or indirectly toat least one modifying group such that the compound modulates theaggregation of natural amyloid proteins or peptides when contacted withthe natural amyloidogenic proteins or peptides. Preferably, the compoundinhibits aggregation of natural amyloidogenic proteins or peptides whencontacted with the natural amyloidogenic proteins or peptides. Theamyloidogenic protein, or peptide fragment thereof, can be, for example,selected from the group consisting of transthyretin (TTR), prion protein(PrP), islet amyloid polypeptide (IAPP), atrial natriuretic factor(ANF), kappa light chain, lambda light chain, amyloid A, procalcitonin,cystatin C, β2 microglobulin, ApoA-I, gelsolin, procalcitonin,calcitonin, fibrinogen and lysozyme.

[0009] In the most preferred embodiment of the invention, the compoundmodulates the aggregation of natural β-AP. The invention provides aβ-amyloid peptide compound comprising a formula:

[0010] wherein Xaa is a β-amyloid peptide having an amino-terminal aminoacid residue corresponding to position 668 of β-amyloid precursorprotein-770 (APP-770) or to a residue carboxy-terminal to position 668of APP-770, A is a modifying group attached directly or indirectly tothe β-amyloid peptide of the compound such that the compound inhibitsaggregation of natural β-amyloid peptides when contacted with thenatural β-amyloid peptides, and n is an integer selected such that thecompound inhibits aggregation of natural β-amyloid peptides whencontacted with the natural β-amyloid peptides.

[0011] In one embodiment, at least one A group is attached directly orindirectly to the amino terminus of the β-amyloid peptide of thecompound. In another embodiment, at least one A group is attacheddirectly or indirectly to the carboxy terminus of the β-amyloid peptideof the compound. In yet another embodiment, at least one A group isattached directly or indirectly to a side chain of at least one aminoacid residue of the β-amyloid peptide of the compound.

[0012] The invention also provides a β-amyloid modulator compoundcomprising an AP aggregation core domain (ACD) coupled directly orindirectly to at least one modifying group (MG) such that the compoundmodulates the aggregation or inhibits the neurotoxicity of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides.Preferably, the Aβ aggregation core domain is modeled after a subregionof natural β-amyloid peptide between 3 and 10 amino acids in length.

[0013] The invention also provides β-amyloid modulator compoundcomprising a formula:

[0014] wherein Xaa₁, Xaa₂ and Xaa₃ are each amino acid structures and atleast two of Xaa₁, Xaa₂ and Xaa₃ are, independently, selected from thegroup consisting of a leucine structure, a phenylalanine structure and avaline structure;

[0015] Y, which may or may not be present, is a peptidic structurehaving the formula (Xaa)_(a), wherein Xaa is any amino acid structureand a is an integer from 1 to 15;

[0016] Z, which may or may not be present, is a peptidic structurehaving the formula (Xaa)_(b), wherein Xaa is any amino acid structureand b is an integer from 1 to 15; and

[0017] A is a modifying group attached directly or indirectly to thecompound and n is an integer;

[0018] Xaa₁, Xaa₂, Xaa₃, Y, Z, A and n being selected such that thecompound modulates the aggregation or inhibits the neurotoxicity ofnatural β-amyloid peptides when contacted with the natural β-amyloidpeptides. In a preferred embodiment, Xaa₁ and Xaa₂ are eachphenylalanine structures. In another preferred embodiment Xaa₂ and Xaa₃are each phenylalanine structures.

[0019] The invention further provides a β-amyloid modulator compoundcomprising a formula:

[0020] wherein

[0021] Xaa₁ and Xaa₃ are amino acid structures;

[0022] Xaa₂ is a valine structure;

[0023] Xaa₄ is a phenylalanine structure;

[0024] Y, which may or may not be present, is a peptidic structurehaving the formula (Xaa)_(a), wherein Xaa is any amino acid structureand a is an integer from 1 to 15;

[0025] Z, which may or may not be present, is a peptidic structurehaving the formula (Xaa)_(b), wherein Xaa is any amino acid structureand b is an integer from 1 to 15; and

[0026] A is a modifying group attached directly or indirectly to thecompound and n is an integer;

[0027] Xaa₁, Xaa₃, Y, Z, A and n being selected such that the compoundmodulates the aggregation or inhibits the neurotoxicity of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides.In a preferred embodiment, Xaa₁ is a leucine structure and Xaa₃ isphenylalanine structure.

[0028] The invention still further provides a compound comprising theformula:

A-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-B

[0029] wherein

[0030] Xaa1 is a histidine structure;

[0031] Xaa2 is a glutamine structure;

[0032] Xaa3 is a lysine structure;

[0033] Xaa4 is a leucine structure;

[0034] Xaa5 is a valine structure;

[0035] Xaa6 is a phenylalanine structure;

[0036] Xaa7 is a phenylalanine structure;

[0037] Xaa8 is an alanine structure;

[0038] A and B are modifying groups attached directly or indirectly tothe amino terminus and carboxy terminus, respectively, of the compound;

[0039] and wherein Xaa₁-Xaa₂-Xaa₃, Xaa₁-Xaa₂ or Xaa₁ may or may not bepresent;

[0040] Xaa₈ may or may not be present; and

[0041] at least one of A and B is present.

[0042] The invention still further provides a β-amyloid modulatorcompound comprising a modifying group attached directly or indirectly toa peptidic structure, wherein the peptidic structure comprises aminoacid structures having an amino acid sequence selected from the groupconsisting of His-Gln-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: 5),His-Gln-Lys-Leu-Val-Phe-Phe (SEQ ID NO: 6), Gln-Lys-Leu-Val-Phe-Phe-Ala(SEQ ID NO: 7), Gln-Lys-Leu-Val-Phe-Phe (SEQ ID NO: 8),Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: 9), Lys-Leu-Val-Phe-Phe (SEQ ID NO:10), Leu-Val-Phe-Phe-Ala (SEQ ID NO: 11), Leu-Val-Phe-Phe (SEQ ID NO:12), Leu-Ala-Phe-Phe-Ala (SEQ ID NO: 13), Val-Phe-Phe (SEQ ID NO: 19),Phe-Phe-Ala (SEQ ID NO: 20), Phe-Phe-Val-Leu-Ala (SEQ ID NO: 21),Leu-Val-Phe-Phe-Lys (SEQ ID NO: 22), Leu-Val-Iodotyrosine-Phe-Ala (SEQID NO: 23), Val-Phe-Phe-Ala (SEQ ID NO: 24), Ala-Val-Phe-Phe-Ala (SEQ IDNO: 25), Leu-Val-Phe-Iodotyrosine-Ala (SEQ ID NO: 26),Leu-Val-Phe-Phe-Ala-Glu (SEQ ID NO: 27), Phe-Phe-Val-Leu (SEQ ID NO:28), Phe-Lys-Phe-Val-Leu (SEQ ID NO: 29), Lys-Leu-Val-Ala-Phe (SEQ IDNO: 30), Lys-Leu-Val-Phe-Phe-βAla (SEQ ID NO: 31) andLeu-Val-Phe-Phe-DAla (SEQ ID NO: 32).

[0043] In the compounds of the invention comprising a modifying group,preferably the modifying group comprises a cyclic, heterocyclic orpolycyclic group. Preferred modifying groups contains a cis-decalingroup, such as a cholanoyl structure. Preferred modifying groups includea cholyl group, a biotin-containing group, adiethylene-triaminepentaacetyl group, a (−)-menthoxyacetyl group, afluorescein-containing group or an N-acetylneuraminyl group.

[0044] The compounds of the invention can be further modified, forexample to alter a pharmacokinetic property of the compound or to labelthe compound with a detectable substance. Preferred radioactive labelsare radioactive iodine or technetium.

[0045] The invention also provides a β-amyloid modulator which inhibitsaggregation of natural β-amyloid peptides when contacted with a molarexcess amount of natural β-amyloid peptides.

[0046] The invention also provides a β-amyloid peptide compoundcomprising an amino acid sequence having at least one amino aciddeletion compared to βAP₁₋₃₉, such that the compound inhibitsaggregation of natural β-amyloid peptides when contacted with thenatural β-amyloid peptides. In one embodiment, the compound has at leastone internal amino acid deleted compared to βAP₆₋₃₉. In anotherembodiment, the compound has at least one N-terminal amino acid deletedcompared to βAP₁₋₃₉. In yet another embodiment, the compound has atleast one C-terminal amino acid deleted compared to βAP₁₋₃₉. Preferredcompounds include βAP₆₋₂₀ (SEQ ID NO: 13), βAP₁₆₋₂₀ (SEQ ID NO: 14),βAP_(1-20, 26-40) (SEQ ID NO: 15) and EEVVHHHHQQ-βAP₁₆₋₄₀ (SEQ ID NO:16).

[0047] The compounds of the invention can be formulated intopharmaceutical compositions comprising the compound and apharmaceutically acceptable carrier. The compounds can also be used inthe manufacture of a medicament for the diagnosis or treatment of anamyloidogenic disease.

[0048] Another aspect of the invention pertains to diagnostic andtreatment methods using the compounds of the invention. The inventionprovides a method for inhibiting aggregation of natural β-amyloidpeptides, comprising contacting the natural β-amyloid peptides with acompound of the invention such that aggregation of the natural β-amyloidpeptides is inhibited. The invention also provides a method forinhibiting neurotoxicity of natural β-amyloid peptides, comprisingcontacting the natural β-amyloid peptides with a compound of theinvention such that neurotoxicity of the natural β-amyloid peptides isinhibited.

[0049] In another embodiment, the invention provides a method fordetecting the presence or absence of natural β-amyloid peptides in abiological sample, comprising contacting a biological sample with acompound of the invention and detecting the compound bound to naturalβ-amyloid peptides to thereby detect the presence or absence of naturalβ-amyloid peptides in the biological sample. In one embodiment, theβ-amyloid modulator compound and the biological sample are contacted invitro. In another embodiment, the β-amyloid modulator compound iscontacted with the biological sample by administering the β-amyloidmodulator compound to a subject. For in vivo administration, preferablythe compound is labeled with radioactive technetium or radioactiveiodine.

[0050] In another embodiment, the invention provides a method fordetecting natural β-amyloid peptides to facilitate diagnosis of aβ-amyloidogenic disease, comprising contacting a biological sample witha compound of the invention and detecting the compound bound to naturalβ-amyloid peptides to facilitate diagnosis of a β-amyloidogenic disease.In one embodiment, the β-amyloid modulator compound and the biologicalsample are contacted in vitro. In another embodiment, the β-amyloidmodulator compound is contacted with the biological sample byadministering the β-amyloid modulator compound to a subject. For in vivoadministration, preferably the compound is labeled with radioactivetechnetium or radioactive iodine. Preferably, the method facilitatesdiagnosis of Alzheimer's disease.

[0051] The invention also provides a method for treating a subject for adisorder associated with amyloidosis, comprising administering to thesubject a therapeutically or prophylactically effective amount of acompound of the invention such that the subject is treated for adisorder associated with amyloidosis. The method can be used to treatdisorders is selected, for example, from the group consisting offamilial amyloid polyneuropathy (Portuguese, Japanese and Swedishtypes), familial amyloid cardiomyopathy (Danish:type), isolated cardiacamyloid, systemic senile amyloidosis, scrapie, bovine spongiformencephalopathy, Creutzfeldt-Jakob disease,Gerstmann-Straussler-Scheinker syndrome, adult onset diabetes,insulinoma, isolated atrial amyloidosis, idiopathic (primary)amyloidosis, myeloma or macroglobulinemia-associated amyloidosis,primary localized cutaneous nodular amyloidosis associated withSjogren's syndrome, reactive (secondary) amyloidosis, familialMediterranean Fever and familial amyloid nephropathy with urticaria anddeafness (Muckle-Wells syndrome), hereditary cerebral hemorrhage withamyloidosis of Icelandic type, amyloidosis associated with long termhemodialysis, hereditary non-neuropathic systemic amyloidosis (familialamyloid polyneuropathy M), familial amyloidosis of Finnish type,amyloidosis associated with medullary carcinoma of the thyroid,fibrinogen-associated hereditary renal amyloidosis andlysozyme-associated hereditary systemic amyloidosis.

[0052] In a preferred embodiment, the invention provides a method fortreating a subject for a disorder associated with β-amyloidosis,comprising administering to the subject a therapeutically orprophylactically effective amount of a compound of the invention suchthat the subject is treated for a disorder associated withβ-amyloidosis. Preferably the disorder is Alzheimer's disease.

[0053] In yet another embodiment, the invention provides a method fortreating a subject for a disorder associated with β-amyloidosis,comprising administering to the subject a recombinant expression vectorencoding a peptide compound of the invention such that the compound issynthesized in the subject and the subject is treated for a disorderassociated with β-amyloidosis. Preferably, the disorder is Alzheimer'sdisease.

BRIEF DESCRIPTION OF THE DRAWING

[0054]FIG. 1 is a graphic representation of the turbidity of a β-AP₁₋₄₀solution, as measured by optical density at 400 nm, either in theabsence of a β-amyloid modulator or in the presence of the β-amyloidmodulator N-biotinyl-βAP₁₋₄₀ (1%, or 5%).

[0055]FIG. 2 is a schematic representation of compounds which can beused to modify a β-AP or an Aβ aggregation core domain to form aβ-amyloid modulator of the invention.

[0056]FIG. 3 is a graphic representation of the toxicity of Aβ₁₋₄₀aggregates, but not Aβ₁₋₄₀ monomers, to cultured neuronal cells.

[0057]FIG. 4 is a graphic representation of the aggregation of Aβ₁₋₄₀ inthe presence of an equimolar amount of cholyl-Aβ₆₋₂₀ (panel A), a˜2-fold molar excess of cholyl-Aβ₆₋₂₀ (panel B) or a ˜6-fold molarexcess of cholyl-Aβ₆₋₂₀ (panel C) and the corresponding toxicity of theaggregates of panels A, B and C to cultured neuronal cells (panels D, Eand F, respectively).

DETAILED DESCRIPTION OF THE INVENTION

[0058] This invention pertains to compounds, and pharmaceuticalcompositions thereof, that can modulate the aggregation of amyloidogenicproteins and peptides, in particular compounds that can modulate theaggregation of natural β amyloid peptides (β-AP) and inhibit theneurotoxicity of natural β-APs. A compound of the invention thatmodulates aggregation of natural β-AP, referred to hereininterchangeably as a β amyloid modulator compound, a β amyloid modulatoror simply a modulator, alters the aggregation of natural β-AP when themodulator is contacted with natural β-AP. Thus, a compound of theinvention acts to alter the natural aggregation process or rate forβ-AP, thereby disrupting this process. Preferably, the compounds inhibitβ-AP aggregation. Furthermore, the invention provides subregions of theD amyloid peptide that are sufficient, when appropriately modified asdescribed herein, to alter (and preferably inhibit) aggregation ofnatural β amyloid peptides when contacted with the natural β amyloidpeptides. In particular, preferred modulator compounds of the inventionare comprised of a modified form of an Aβ aggregation core domain,modeled after the aforementioned Aβ subregion (as described furtherbelow), which is sufficient to alter (and preferably inhibit) thenatural aggregation process or rate for β-AP. This Aβ aggregation coredomain can comprises as few as three amino acid residues (or derivative,analogues or mimetics thereof). Moreover, while the amino acid sequenceof the Aβ aggregation core domain can directly correspond to an aminoacid sequence found in natural β-AP, it is not essential that the aminoacid sequence directly correspond to a β-AP sequence. Rather, amino acidresidues derived from a preferred subregion of β-AP (a hydrophobicregion centered around positions 17-20) can be rearranged in orderand/or substituted with homologous residues within a modulator compoundof the invention and yet maintain their inhibitory activity (describedfurther below).

[0059] The β amyloid modulator compounds of the invention can beselected based upon their ability to inhibit the aggregation of naturalβ-AP in vitro and/or inhibit the neurotoxicity of natural β-AP fibrilsfor cultured cells (using assays described herein). Accordingly, thepreferred modulator compounds inhibit the aggregation of natural β-APand/or inhibit the neurotoxicity of natural β-AP. However, modulatorcompounds selected based on one or both of these properties may haveadditional properties in vivo that may be beneficial in the treatment ofamyloidosis. For example, the modulator compound may interfere withprocessing of natural β-AP (either by direct or indirect proteaseinhibition) or by modulation of processes that produce toxic β-AP, orother APP fragments, in vivo. Alternatively, modulator compounds may beselected based on these latter properties, rather than inhibition of Aβaggregation in vitro. Moreover, modulator compounds of the inventionthat are selected based upon their interaction with natural β-AP alsomay interact with APP or with other APP fragments.

[0060] As used herein, a “modulator” of β-amyloid aggregation isintended to refer to an agent that, when contacted with natural βamyloid peptides, alters the aggregation of the natural β amyloidpeptides. The term “aggregation of β amyloid peptides” refers to aprocess whereby the peptides associate with each other to form amultimeric, largely insoluble complex. The term “aggregation” further isintended to encompass β amyloid fibril formation and also encompassesβ-amyloid plaques.

[0061] The terms “natural β-amyloid peptide”, “natural β-AP” and“natural Aβ peptide”, used interchangeably herein, are intended toencompass naturally occurring proteolytic cleavage products of the βamyloid precursor protein (APP) which are involved in β-AP aggregationand β-amyloidosis. These natural peptides include β-amyloid peptideshaving 39-43 amino acids (i.e., Aβ₁₋₃₉, Aβ₁₋₄₀, Aβ₁₋₄₁, Aβ₁₋₄₂ andAβ₁₋₄₃). The amino-terminal amino acid residue of natural β-APcorresponds to the aspartic acid residue at position 672 of the 770amino acid residue form of the amyloid precursor protein (“APP-770”).The 43 amino acid long form of natural β-AP has the amino acid sequenceDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIAT (also shown in SEQ ID NO:1), whereas the shorter forms have 1-4 amino acid residues deleted fromthe carboxy-terminal end. The amino acid sequence of APP-770 fromposition 672 (i.e., the amino-terminus of natural β-AP) to itsC-terminal end (103 amino acids) is shown in SEQ ID NO: 2. The preferredform of natural β-AP for use in the aggregation assays described hereinis Aβ₁₋₄₀.

[0062] In the presence of a modulator of the invention, aggregation ofnatural β amyloid peptides is “altered” or “modulated”. The variousforms of the term “alteration” or “modulation” are intended to encompassboth inhibition of β-AP aggregation and promotion of β-AP aggregation.Aggregation of natural β-AP is “inhibited” in the presence of themodulator when there is a decrease in the amount and/or rate of β-APaggregation as compared to the amount and/or rate of β-AP aggregation inthe absence of the modulator. The various forms of the term “inhibition”are intended to include both complete and partial inhibition of β-APaggregation. Inhibition of aggregation can be quantitated as the foldincrease in the lag time for aggregation or as the decrease in theoverall plateau level of aggregation (i.e., total amount ofaggregation), using an aggregation assay as described in the Examples.In various embodiments, a modulator of the invention increases the lagtime of aggregation at least 1.2-fold, 1.5-fold, 1.8-fold, 2-fold,2.5-fold, 3-fold, 4-fold or 5-fold. In various other embodiments, amodulator of the invention inhibits the plateau level of aggregation atleast 10%, 20%, 30%, 40%, 50%, 75% or 100%.

[0063] A modulator which inhibits β-AP aggregation (an “inhibitorymodulator compound”) can be used to prevent or delay the onset ofβ-amyloid deposition. Moreover, as demonstrated in Example 10,inhibitory modulator compounds of the invention inhibit the formationand/or activity of neurotoxic aggregates of natural Aβ peptide (i.e.,the inhibitory compounds can be used to inhibit the neurotoxicity ofβ-AP). Still further, also as demonstrated in Example 10, the inhibitorycompounds of the invention can be used to reduce the neurotoxicity ofpreformed β-AP aggregates, indicating that the inhibitory modulators caneither bind to preformed Aβ fibrils or soluble aggregate and modulatetheir inherent neurotoxicity or that the modulators can perturb theequilibrium between monomeric and aggregated forms of β-AP in favor ofthe non-neurotoxic form.

[0064] Alternatively, in another embodiment, a modulator compound of theinvention promotes the aggregation of natural Aβ peptides. The variousforms of the term “promotion” refer to an increase in the amount and/orrate of β-AP aggregation in the presence of the modulator, as comparedto the amount and/or rate of β-AP aggregation in the absence of themodulator. Such a compound which promotes Aβ aggregation is referred toas a stimulatory modulator compound. Stimulatory modulator compounds maybe useful for sequestering β-amyloid peptides, for example in abiological compartment where aggregation of β-AP may not be deleteriousto thereby deplete β-AP from a biological compartment where aggregationof β-AP is deleterious. Moreover, stimulatory modulator compounds can beused to promote Aβ aggregation in in vitro aggregation assays (e.g.,assays such as those described in the Examples), for example inscreening assays for test compounds that can then inhibit or reversethis Aβ aggregation (i.e., a stimulatory modulator compound can act as a“seed” to promote the formation of Aβ aggregates).

[0065] In a preferred embodiment, the modulators of the invention arecapable of altering β-AP aggregation when contacted with a molar excessamount of natural β-AP. A “molar excess amount of natural β-AP” refersto a concentration of natural β-AP, in moles, that is greater than theconcentration, in moles, of the modulator. For example, if the modulatorand β-AP are both present at a concentration of 1 μM, they are said tobe “equimolar”, whereas if the modulator is present at a concentrationof 1 μM and the β-AP is present at a concentration of 5 μM, the β-AP issaid to be present at a 5-fold molar excess amount compared to themodulator. In preferred embodiments, a modulator of the invention iseffective at altering natural β-AP aggregation when the natural β-AP ispresent at at least a 2-fold, 3-fold or 5-fold molar excess compared tothe concentration of the modulator. In other embodiments, the modulatoris effective at altering β-AP aggregation when the natural β-AP ispresent at at least a 10-fold, 20-fold, 33-fold, 50-fold, 100-fold,500-fold or 1000-fold molar excess compared to the concentration of themodulator.

[0066] Various additional aspects of the modulators of the invention,and the uses thereof, are described in further detail in the followingsubsections.

[0067] I. Modulator Compounds

[0068] In one embodiment, a modulator of the invention comprises aβ-amyloid peptide compound comprising the formula:

[0069] wherein Xaa is a β-amyloid peptide, A is a modulating groupattached directly or indirectly to the β-amyloid peptide of the compoundsuch that the compound inhibits aggregation of natural β-amyloidpeptides when contacted with the natural β-amyloid peptides, and n is aninteger selected such that the compound inhibits aggregation of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides.

[0070] Preferably, β-amyloid peptide of the compound has anamino-terminal amino acid residue corresponding to position 668 ofβ-amyloid precursor protein-770 (APP-770) or to a residuecarboxy-terminal to position 668 of APP-770. The amino acid sequence ofAPP-770 from position 668 to position 770 (i.e., the carboxy terminus)is shown below and in SEQ ID NO: 2:EVKMDAEFRHDSGYEVHHQKiLVFFAEDVGSNKGAIIGLMVGGVVIATVIVITLVMLKKKQYTSIIIHGVVEVDAAVTPEERLILSKMQQNGYENPTYKF FEQMQN

[0071] More preferably, the amino-terminal amino acid residue of theβ-amyloid peptide corresponds to position 672 of APP-770 (position 5 ofthe amino acid sequence of SEQ ID NO: 2) or to a residuecarboxy-terminal to position 672 of APP-770. Although the β-amyloidpeptide of the compound may encompass the 103 amino acid residuescorresponding to positions 668-770 of APP-770, preferably the peptide isbetween 6 and 60 amino acids in length, more preferably between 10 and43 amino acids in length and even more preferably between 10 and 25amino acid residues in length.

[0072] As used herein, the term “β amyloid peptide”, as used in amodulator of the invention is intended to encompass peptides having anamino acid sequence identical to that of the natural sequence in APP, aswell as peptides having acceptable amino acid substitutions from thenatural sequence. Acceptable amino acid substitutions are those that donot affect the ability of the peptide to alter natural β-AP aggregation.Moreover, particular amino acid substitutions may further contribute tothe ability of the peptide to alter natural β-AP aggregation and/or mayconfer additional beneficial properties on the peptide (e.g., increasedsolubility, reduced association with other amyloid proteins, etc.). Forexample, substitution of hydrophobic amino acid residues for the twophenylalanine residues at positions 19 and 20 of natural β-AP (positions19 and 20 of the amino acid sequence shown in SEQ ID NO: 1) may furthercontribute to the ability of the peptide to alter β-AP aggregation (seeHilbich, C. (1992) J. Mol. Biol. 228:460-473). Thus, in one embodiment,the M-AP of the compound consists of the amino acid sequence shown belowand in SEQ ID NO: 3:

[0073] DAEFRHDSGYEVHHQKLV(Xaa₁₉)(Xaa₂₀)AEDVGSNKGAIIGLMVGGVVIAT

[0074] (or an amino-terminal or carboxy-terminal deletion thereof),wherein Xaa is a hydrophobic amino acid. Examples of hydrophobic aminoacids are isoleucine, leucine, threonine, serine, alanine, valine orglycine. Preferably, F₁₉F₂₀ is substituted with T₁₉T₂₀ or G₁₉I₂₀.

[0075] Other suitable amino acid substitutions include replacement ofamino acids in the human peptide with the corresponding amino acids ofthe rodent β-AP peptide. The three amino acid residues that differbetween human and rat β-AP are at positions 5, 10 and 13 of the aminoacid sequence shown in SEQ ID NOs: 1 and 3. A human β-AP having thehuman to rodent substitutions Arg₅ to Gly, Tyr₁₀ to Phe and His₁₃ to Arghas been shown to retain the properties of the human peptide (seeFraser, P. E. et al. (1992) Biochemistry 31:10716-10723; and Hilbich, C.et al. (1991) Eur. J. Biochem. 201:61-69). Accordingly, a human β-APhaving rodent β-AP a.a. substitutions is suitable for use in a modulatorof the invention.

[0076] Other possible β-AP amino acid substitutions are described inHilbich, C. et al. (1991) J. Mol. Biol. 218:149-163; and Hilbich, C.(1992) J. Mol. Biol. 228:460-473. Moreover, amino acid substitutionsthat affect the ability of β-AP to associate with other proteins can beintroduced. For example, one or more amino acid substitutions thatreduce the ability of β-AP to associate with the serpin enzyme complex(SEC) receptor, α1-antichymotrypsin (ACT) and/or apolipoprotein E (ApoE)can be introduced. A preferred substitution for reducing binding to theSEC receptor is L₃₄M₃₅ to A₃₄A₃₅ (at positions 34 and 35 of the aminoacid sequences shown in SEQ ID NOs: 1 and 3). A preferred substitutionfor reducing binding to ACT is S₈ to A₈ (at position 8 of the amino acidsequences shown in SEQ ID NOs: 1 and 3).

[0077] Alternative to β-AP amino acid substitutions described herein orknown in the art, a modulator composed, at least in part, of an aminoacid-substituted β amyloid peptide can be prepared by standardtechniques and tested for the ability to alter β-AP aggregation using anaggregation assay described herein. To retain the properties of theoriginal modulator, preferably conservative amino acid substitutions aremade at one or more amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art,including basic side chains (e.g., lysine, arginine, histidine), acidicside chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),β-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Accordingly, a modulator composed of a β amyloid peptidehaving an amino acid sequence that is mutated from that of the wild-typesequence in APP-770 yet which still retains the ability to alter naturalβ-AP aggregation is within the scope of the invention.

[0078] As used herein, the term “β amyloid peptide” is further intendedto include peptide analogues or peptide derivatives or peptidomimeticsthat retain the ability to alter natural β-AP aggregation as describedherein. For example, a β amyloid peptide of a modulator of the inventionmay be modified to increase its stability, bioavailability, solubility,etc. The terms “peptide analogue”, “peptide derivative” and“peptidomimetic” as used herein are intended to include molecules whichmimic the chemical structure of a peptide and retain the functionalproperties of the peptide. Approaches to designing peptide analogs areknown in the art. For example, see Farmer, P. S. in Drug Design (E. J.Ariens, ed.) Academic Press, New York, 1980, vol. 10, pp. 119-143; Ball.J. B. and Alewood, P. F. (1990) J. Mol. Recognition 3:55; Morgan, B. A.and Gainor, J. A. (1989) Ann. Rep. Med. Chem. 24:243; and Freidinger, R.M. (1989) Trends Pharmacol. Sci. 10:270. Examples of peptide analogues,derivatives and peptidomimetics include peptides substituted with one ormore benzodiazepine molecules (see e.g., James, G. L. et al. (1993)Science 260:1937-1942), peptides with methylated amide linkages and“retro-inverso” peptides (see U.S. Pat. No. 4,522,752 by Sisto). Peptideanalogues, peptide derivatives and peptidomimetic are described infurther detail below with regard to compounds comprising an Aβaggregation core domain.

[0079] In a modulator of the invention having the formula shown above, amodulating group (“A”) is attached directly or indirectly to theβ-amyloid peptide of the modulator (As used herein, the term “modulatinggroup” and “modifying group” are used interchangeably to describe achemical group directly or indirectly attached to an AP derived peptidicstructure). For example, the modulating group can be directly attachedby covalent coupling to the β-amyloid peptide or the modulating groupcan be attached indirectly by a stable non-covalent association. In oneembodiment of the invention, the modulating group is attached to theamino-terminus of the β-amyloid peptide of the modulator. Accordingly,the modulator can comprise a compound having a formula:

[0080] Alternatively, in another embodiment of the invention, themodulating group is attached to the carboxy-terminus of the β-amyloidpeptide of the modulator. Accordingly, the modulator can comprise acompound having a formula:

[0081] In yet another embodiment, the modulating group is attached tothe side chain of at least one amino acid residues of the β-amyloidpeptide of the compound (e.g., through the epsilon amino group of alysyl residue(s), through the carboxyl group of an aspartic acidresidue(s) or a glutamic acid residue(s), through a hydroxy group of atyrosyl residue(s), a serine residue(s) or a threonine residue(s) orother suitable reactive group on an amino acid side chain).

[0082] The modulating group is selected such that the compound inhibitsaggregation of natural β-amyloid peptides when contacted with thenatural β-amyloid peptides. Accordingly, since the β-AP peptide of thecompound is modified from its natural state, the modulating group “A” asused herein is not intended to include hydrogen. In a preferredembodiment, the modulating group is a biotin compound of the formula:

[0083] wherein X₁-X₃ are each independently selected from the groupconsisting of S, O and NR₂, wherein R₂ is hydrogen, or an aryl, loweralkyl, alkenyl or alkynyl moiety; W is ═O or NR₂; R₁ is a loweralkylenyl moiety and Y is a direct bond or a spacer molecule selectedfor its ability to react with a target group on a β-AP. At least one ofX₁—X₃ or W is an NR₂ group.

[0084] The term “aryl” is intended to include aromatic moietiescontaining substituted or unsubstituted ring(s), e.g., benzyl, napthyl,etc. Other more complex fused ring moieties also are intended to beincluded.

[0085] The term “lower alkyl or alkylenyl moiety” refers to a saturated,straight or branched chain (or combination thereof) hydrocarboncontaining 1 to about 6 carbon atoms, more preferably from 1 to 3 carbonatoms. The terms “lower alkenyl moiety” and “lower alkynyl moiety” referto unsaturated hydrocarbons containing 1 to about 6 carbon atoms, morepreferably 1 to 3 carbon atoms. Preferably, R₂ contains 1 to 3 carbonatoms. Preferably, R₁ contains 4 carbon atoms.

[0086] The spacer molecule (Y) can be, for example, a lower alkyl groupor a linker peptide, and is preferably selected for its ability to linkwith a free amino group (e.g., the α-amino group at the amino-terminusof a β-AP). Thus, in a preferred embodiment, the biotin compoundmodifies the amino-terminus of a β-amyloid peptide.

[0087] Additional suitable modulating groups may include other cyclicand heterocyclic compounds and other compounds having similar steric“bulk”. Non-limiting examples of compounds which can be used to modify aβ-AP are shown schematically in FIG. 2, and include N-acetylneuraminicacid, cholic acid, trans-4-cotininecarboxylic acid,2-imino-1-imidazolidineacetic acid, (S)-(−)-indoline-2-carboxylic acid,(−)-menthoxyacetic acid, 2-norbornaneacetic acid,γ-oxo-5-acenaphthenebutyric acid, (−)-2-oxo-4-thiazolidinecarboxylicacid, tetrahydro-3-furoic acid, 2-iminobiotin-N-hydroxysuccinimideester, diethylenetriaminepentaacetic dianhydride, 4-morpholinecarbonylchloride, 2-thiopheneacetyl chloride, 2-thiophenesulfonyl chloride,5-(and 6-)-carboxyfluorescein (succinimidyl ester), fluoresceinisothiocyanate, and acetic acid (or derivatives thereof). Suitablemodulating groups are described further in subsection II below.

[0088] In a modulator of the invention, a single modulating group may beattached to a amyloid peptide (e.g., n=1 in the formula shown above) ormultiple modulating groups may be attached to the peptide. The number ofmodulating groups is selected such that the compound inhibitsaggregation of natural β-amyloid peptides when contacted with thenatural β-amyloid peptides. However, n preferably is an integer between1 and 60, more preferably between 1 and 30 and even more preferablybetween 1 and 10 or 1 and 5.

[0089] In another embodiment, a β-amyloid modulator compound of theinvention comprises an Aβ aggregation core domain (abbreviated as ACD)coupled directly or indirectly to a modifying group such that thecompound modulates the aggregation or inhibits the neurotoxicity ofnatural β-amyloid peptides when contacted with the natural β-amyloidpeptides. As used herein, an “AP aggregation core domain” is intended torefer to a structure that is modeled after a subregion of a naturalβ-amyloid peptide which is sufficient to modulate aggregation of naturalβ-APs when this subregion of the natural G-AP is appropriately modifiedas described herein (e.g., modified at the amino-terminus). The term“subregion of a natural β-amyloid peptide” is intended to includeamino-terminal and/or carboxy-terminal deletions of natural G-AP. Theterm “subregion of natural β-AP” is not intended to include full-lengthnatural G-AP (i.e., “subregion” does not include Aβ₁₋₃₉, Aβ₁₋₄₀, Aβ₁₋₄₁,Aβ₁₋₄₂ and Aβ₁₋₄₃).

[0090] Although not intending to be limited by mechanism, the ACD of themodulators of the invention is thought to confer a specific targetingfunction on the compound that allows the compound to recognize andspecifically interact with natural β-AP. Preferably, the ACD is modeledafter a subregion of natural β-AP that is less than 15 amino acids inlength and more preferably is between 3-10 amino acids in length. Invarious embodiments, the ACD is modeled after a subregion of β-AP thatis 10, 9, 8, 7, 6, 5, 4 or 3 amino acids in length. In one embodiment,the subregion of β-AP upon which the ACD is modeled is an internal orcarboxy-terminal region of β-AP (i.e., downstream of the amino-terminusat amino acid position 1). In another embodiment, the ACD is modeledafter a subregion of β-AP that is hydrophobic. In certain specificembodiments, the term Aβ aggregation core domain specifically excludesβ-AP subregions corresponding to amino acid positions 1-15 (Aβ₁₋₁₅),6-20.(Aβ₆₋₂₀) and 16-40 (Aβ₁₆₋₄₀).

[0091] An Aβ aggregation core domain can be comprised of amino acidresidues linked by peptide bonds. That is, the ACD can be a peptidecorresponding to a subregion of β-AP. Alternatively, an Aβ aggregationcore domain can be modeled after the natural Aβ peptide region but maybe comprised of a peptide analogue, peptide derivative or peptidomimeticcompound, or other similar compounds which mimics the structure andfunction of the natural peptide. Accordingly, as used herein, an “Aβaggregation core domain” is intended to include peptides, peptideanalogues, peptide derivatives and peptidomimetic compounds which, whenappropriately modified, retain the aggregation modulatory activity ofthe modified natural Aβ peptide subregion. Such structures that aredesigned based upon the amino acid sequence are referred to herein as“AP derived peptidic structures.” Approaches to designing peptideanalogues, derivatives and mimetics are known in the art. For example,see Farmer, P. S. in Drug Design (E. J. Ariens, ed.) Academic Press, NewYork, 1980, vol. 10, pp. 119-143; Ball. J. B. and Alewood, P. F. (1990)J Mol. Recognition 3:55; Morgan, B. A. and Gainor, J. A. (1989) Ann.Rep. Med. Chem. 24:243; and Freidinger, R. M. (1989) Trends Pharmacol.Sci. 10:270. See also Sawyer, T. K. (1995) “Peptidomimetic Design andChemical Approaches to Peptide Metabolism” in Taylor, M. D. and Amidon,G. L. (eds.) Peptide-Based Drug Design: Controlling Transport andMetabolism, Chapter 17; Smith, A. B. 3rd, et al. (1995) J. Am. Chem.Soc. 117:11113-11123; Smith, A. B. 3rd, et al. (1994) J. Am. Chem. Soc.116:9947-9962; and Hirschman, R., et al. (1993) J. Am. Chem. Soc.115:12550-12568.

[0092] As used herein, a “derivative” of a compound X (e.g., a peptideor amino acid) refers to a form of X in which one or more reactiongroups on the compound have been derivatized with a substituent group.Examples of peptide derivatives include peptides in which an amino acidside chain, the peptide backbone, or the amino- or carboxy-terminus hasbeen derivatized (e.g., peptidic compounds with methylated amidelinkages). As used herein an “analogue” of a compound X refers to acompound which retains chemical structures of X necessary for functionalactivity of X yet which also contains certain chemical structures whichdiffer from X. An examples of an analogue of a naturally-occurringpeptide is a peptides which includes one or more non-naturally-occurringamino acids. As used herein, a “mimetic” of a compound X refers to acompound in which chemical structures of X necessary for functionalactivity of X have been replaced with other chemical structures whichmimic the conformation of X. Examples of peptidomimetics includepeptidic compounds in which the peptide backbone is substituted with oneor more benzodiazepine molecules (see e.g., James, G. L. et al. (1993)Science 260:1937-1942), peptides in which all L-amino acids aresubstituted with the corresponding D-amino acids and “retro-inverso”peptides (see U.S. Pat. No. 4,522,752 by Sisto), described furtherbelow.

[0093] The term mimetic, and in particular, peptidomimetic, is intendedto include isosteres. The term “isostere” as used herein is intended toinclude a chemical structure that can be substituted for a secondchemical structure because the steric conformation of the firststructure fits a binding site specific for the second structure. Theterm specifically includes peptide back-bone modifications (i.e., amidebond mimetics) well known to those skilled in the art. Suchmodifications include modifications of the amide nitrogen, the α-carbon,amide carbonyl, complete replacement of the amide bond, extensions,deletions or backbone crosslinks. Several peptide backbone modificationsare known, including ψ[CH₂S], ψ[CH₂NH], ψ[CSNH₂], ψ[NHCO], ψ[COCH₂], andψ[(E) or (Z) CH═CH]. In the nomenclature used above, ψ indicates theabsence of an amide bond. The structure that replaces the amide group isspecified within the brackets. Other examples of isosteres includepeptides substituted with one or more benzodiazepine molecules (seee.g., James, G. L. et al. (1993) Science 260:1937-1942)

[0094] Other possible modifications include an N-alkyl (or aryl)substitution (ψ[CONR]), backbone crosslinking to construct lactams andother cyclic structures, substitution of all D-amino acids for allL-amino acids within the compound (“inverso” compounds) or retro-inversoamino acid incorporation (ψ[NHCO]). By “inverso” is meant replacingL-amino acids of a sequence with D-amino acids, and by “retro-inverso”or “enantio-retro” is meant reversing the sequence of the amino acids(“retro”) and replacing the L-amino acids with D-amino acids. Forexample, if the parent peptide is Thr-Ala-Tyr, the retro modified formis Tyr-Ala-Thr, the inverso form is thr-ala-tyr, and the retro-inversoform is tyr-ala-thr (lower case letters refer to D-amino acids).Compared to the parent peptide, a retro-inverso peptide has a reversedbackbone while retaining substantially the original spatial conformationof the side chains, resulting in a retro-inverso isomer with a topologythat closely resembles the parent peptide. See Goodman et al.“Perspectives in Peptide Chemistry” pp. 283-294 (1981). See also U.S.Pat. No. 4,522,752 by Sisto for further description of “retro-inverso”peptides.

[0095] Other derivatives of the modulator compounds of the inventioninclude C-terminal hydroxymethyl derivatives, O-modified derivatives(e.g., C-terminal hydroxymethyl benzyl ether), N-terminally modifiedderivatives including substituted amides such as alkylamides andhydrazides and compounds in which a C-terminal phenylalanine residue isreplaced with a phenethylamide analogue (e.g., Val-Phe-phenethylamide asan analogue of the tripeptide Val-Phe-Phe).

[0096] In a preferred embodiment, the ACD of the modulator is modeledafter the subregion of β-AP encompassing amino acid positions 17-20(i.e., Leu-Val-Phe-Phe; SEQ ID NO: 12). As described further in Examples7, 8 and 9, peptide subregions of Aβ₁₋₄₀ were prepared, amino-terminallymodified and evaluated for their ability to modulate aggregation ofnatural β-amyloid peptides. One subregion that was effective atinhibiting aggregation was AP₆₋₂₀ (i.e., amino acid residues 6-20 of thenatural Aβ₁₋₄₀ peptide, the amino acid sequence of which is shown in SEQID NO: 4). Amino acid residues were serially deleted from theamino-terminus or carboxy terminus of this subregion to furtherdelineate a minimal subregion that was sufficient for aggregationinhibitory activity. This process defined Aβ₁₇₋₂₀ (i.e., amino acidresidues 17-20 of the natural Aβ₁₋₄₀ peptide) as a minimal subregionthat, when appropriately modified, is sufficient for aggregationinhibitory activity. Accordingly, an “Aβ aggregation core domain” withina modulator compound of the invention can be modeled after Aβ₁₇₋₂₀. Inone embodiment, the Aβ aggregation core domain comprises Aβ₁₇₋₂₀ itself(i.e., a peptide comprising the amino acid sequenceleucine-valine-phenylalanine-phenylalanine; SEQ ID NO: 12). In otherembodiments, the structure of AP₁₇₋₂₀ is used as a model to design an Aβaggregation core domain having similar structure and function toAβ₁₇₋₂₀. For example, peptidomimetics, derivatives or analogues ofAβ₁₇₋₂₀ (as described above) can be used as an AP aggregation coredomain. In addition to Aβ₁₇₋₂₀, the natural Aβ peptide is likely tocontain other minimal subregions that are sufficient for aggregationinhibitory activity. Such additional minimal subregions can beidentified by the processes described in Examples 7, 8 and 9, wherein a15mer subregion of Aβ₁₋₄₀ is serially deleted from the amino-terminus orcarboxy terminus, the deleted peptides are appropriately modified andthen evaluated for aggregation inhibitory activity.

[0097] One form of the β-amyloid modulator compound comprising an Aβaggregation core domain modeled after Aβ₁₇₋₂₀ coupled directly orindirectly to at least one modifying group has the formula:

[0098] wherein

[0099] Xaa₁ and Xaa₃ are amino acid structures;

[0100] Xaa₂ is a valine structure;

[0101] Xaa₄ is a phenylalanine structure;

[0102] Y, which may or may not be present, is a peptidic structurehaving the formula (Xaa)_(a), wherein Xaa is any amino acid structureand a is an integer from 1 to 15;

[0103] Z, which may or may not be present, is a peptidic structurehaving the formula (Xaa)_(b), wherein Xaa is any amino acid structureand b is an integer from 1 to 15; and

[0104] A is a modifying group attached directly or indirectly to thecompound and n is an integer;

[0105] Xaa₁, Xaa₃, Y, Z, A and n being selected such that the compoundmodulates the aggregation or inhibits the neurotoxicity of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides.

[0106] Preferably, a modulator compound of the above formula inhibitsaggregation of natural β-amyloid peptides when contacted with thenatural β-amyloid peptides and/or inhibits Aβ neurotoxicity.Alternatively, the modulator compound can promote aggregation of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides.The type and number of modifying groups (“A”) coupled to the modulatorare selected such that the compound alters (and preferably inhibits)aggregation of natural β-amyloid peptides when contacted with thenatural β-amyloid peptides. A single modifying group can be coupled tothe modulator (i.e., n=1 in the above formula) or, alternatively,multiple modifying groups can be coupled to the modulator. In variousembodiments, n is an integer between 1 and 60, between 1 and 30, between1 and 10, between 1 and 5 or between 1 and 3. Suitable types ofmodifying groups are described further in subsection II below.

[0107] As demonstrated in Example 9, amino acid positions 18 (Val₁₈) and20 (Phe₂₀) of Aβ₁₇₋₂₀ (corresponding to Xaa₂ and Xaa₄) are particularlyimportant within the core domain for inhibitory activity of themodulator compound. Accordingly, these positions are conserved withinthe core domain in the formula shown above. The terms “valine structure”and “phenylalanine structure” as used in the above formula are intendedto include the natural amino acids, as well as non-naturally-occurringanalogues, derivatives and mimetics of valine and phenylalanine,respectively, (including D-amino acids) which maintain the functionalactivity of the compound. Moreover, although Val₁₈ and Phe₂₀ have animportant functional role, it is possible that Xaa₂ and/or Xaa₄ can besubstituted with other naturally-occurring amino acids that arestructurally related to valine or phenylalanine, respectively, whilestill maintaining the activity of the compound. Thus, the terms “valinestructure” is intended to include conservative amino acid substitutionsthat retain the activity of valine at Xaa₂, and the term “phenylalaninestructure” is intended to include conservative amino acid substitutionsthat retain the activity of phenylalanine at Xaa₄. However, the term“valine structure” is not intended to include threonine.

[0108] In contrast to positions 18 and 20 of Aβ17-20, a Phe to Alasubstitution at position 19 (corresponding to Xaa₃) did not abolish theactivity of the modulator, indicating position 19 may be more amenableto amino acid substitution. In various embodiments of the above formula,positions Xaa₁ and Xaa₃ are any amino acid structure. The term “aminoacid structure” is intended to include natural and non-natural aminoacids as well as analogues, derivatives and mimetics thereof, includingD-amino acids. In a preferred embodiment of the above formula, Xaa₁ is aleucine structure and Xaa₃ is a phenylalanine structure (i.e., modeledafter Leu₁₇ and Phe₁₉, respectively, in the natural AP peptidesequence). The term “leucine structure” is used in the same manner asvaline structure and phenylalanine structure described above.Alternatively, an another embodiment, Xaa₃ is an alanine structure.

[0109] The four amino acid structure ACD of the modulator of the aboveformula can be flanked at the amino-terminal side, carboxy-terminalside, or both, by peptidic structures derived either from the natural Aβpeptide sequence or from non-Aβ sequences. The term “peptidic structure”is intended to include peptide analogues, derivatives and mimeticsthereof, as described above. The peptidic structure is composed of oneor more linked amino acid structures, the type and number of which inthe above formula are variable. For example, in one embodiment, noadditional amino acid structures flank the Xaa₁-Xaa₂-Xaa₃-Xaa₄ coresequence (i.e., Y and Z are absent in the above formula). In anotherembodiment, one or more additional amino acid structures flank only theamino-terminus of the core sequences (i.e., Y is present but Z is absentin the above formula). In yet another embodiment, one or more additionalamino acid structures flank only the carboxy-terminus of the coresequences (i.e., Z is present but Y is absent in the above formula). Thelength of flanking Z or Y sequences also is variable. For example, inone embodiment, a and b are integers from 1 to 15. More preferably, aand b are integers between 1 and 10. Even more preferably, a and b areintegers between 1 and 5. Most preferably, a and b are integers between1 and 3.

[0110] One form of the β-amyloid modulator compound comprising an APaggregation core domain modeled after Aβ₁₇₋20 coupled directly orindirectly to at least one modifying group has the formula:

A-(Y)-Xaa₁-Xaa₂-Xaa₃-Xaa4-(Z)-B

[0111] wherein

[0112] Xaa₁ and Xaa₃ are amino acids or amino acid mimetics;

[0113] Xaa₂ is valine or a valine mimetic

[0114] Xaa₄ is phenylalanine or a phenylalanine mimetic;

[0115] Y, which may or may not be present, is a peptide orpeptidomimetic having the formula (Xaa)_(a), wherein Xaa is any aminoacid or amino acid mimetic and a is an integer from 1 to 15;

[0116] Z, which may or may not be present, is a peptide or-peptidomimetic having the formula (Xaa)_(b), wherein Xaa is any aminoacid or amino acid mimetic and b is an integer from 1 to 15; and

[0117] A and B, at least one of which is present, are modifying groupsattached directly or indirectly to the amino terminus and carboxyterminus, respectively, of the compound;

[0118] Xaa₁, Xaa₃, Y, Z, A and B being selected such that the compoundmodulates the aggregation or inhibits the neurotoxicity of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides.

[0119] In this embodiment, the modulator compound is specificallymodified at either its amino-terminus, its carboxy-terminus, or both.The terminology used in this formula is the same as described above.Suitable modifying groups are described in subsection II below. In oneembodiment, the compound is modified only at its amino terminus (i.e., Bis absent and the compound comprises the formula:A-(Y)-Xaa₁-Xaa₂-Xaa₃-Xaa₄-(Z)). In another embodiment, the compound ismodified only at its carboxy-terminus (i.e., A is absent and thecompound comprises the formula: (Y)-Xaa₁-Xaa₂-Xaa₃-Xaa₄-(Z)-B). In yetanother embodiment, the compound is modified at both its amino- andcarboxy termini (i.e., the compound comprises the formula:A-(Y)-Xaa₁-Xaa₂-Xaa₃-Xaa₄-(Z)-B and both A and B are present). Asdescribed above, the type and number of amino acid structures whichflank the Xaa₁-Xaa₂-Xaa₃-Xaa₄ core sequences in the above formula isvariable. For example, in one embodiment, a and b are integers from 1 to15. More preferably, a and b are integers between 1 and 10. Even morepreferably, a and b are integers between 1 and 5. Most preferably, a andb are integers between 1 and 3.

[0120] As demonstrated in Examples 7, 8 and 9, preferred AP modulatorcompounds of the invention comprise modified forms of Aβ₁₄₋₂₁(His-Gln-Lys-Leu-Val-Phe-Phe-Ala; SEQ ID NO: 5), or amino-terminal orcarboxy-terminal deletions thereof, with a preferred “minimal coreregion” comprising Aβ₁₇₋₂₀. Accordingly, in specific embodiments, theinvention provides compounds comprising the formula:

A-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-B

[0121] wherein

[0122] Xaa1 is a histidine structure;

[0123] Xaa2 is a glutamine structure;

[0124] Xaa3 is a lysine structure;

[0125] Xaa4 is a leucine structure;

[0126] Xaa5 is a valine structure;

[0127] Xaa6 is a phenylalanine structure;

[0128] Xaa7 is a phenylalanine structure;

[0129] Xaa8 is an alanine structure;

[0130] A and B are modifying groups attached directly or indirectly tothe amino terminus and carboxy terminus, respectively, of the compound;

[0131] and wherein

[0132] Xaa₁-Xaa₂-Xaa₃, Xaa₁-Xaa₂ or Xaa₁ may or may not be present;

[0133] Xaa₈ may or may not be present; and

[0134] at least one of A and B is present.

[0135] In one specific embodiment, the compound comprises the formula:A-Xaa₄-Xaa₅-Xaa₆-Xaa₇-B (e.g, a modified form of AP₁₇₋₂₀, comprising anamino acid sequence Leu-Val-Phe-Phe; SEQ ID NO: 12).

[0136] In another specific embodiment, the compound comprises theformula: A-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-B (e.g, a modified form of Aβ₁₇₋₂₁,comprising an amino acid sequence Leu-Val-Phe-Phe-Ala; SEQ ID NO: 11).

[0137] In another specific embodiment, the compound comprises theformula: A-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-B (e.g., a modified form of Aβ₁₆₋₂₀,comprising an amino acid sequence Lys-Leu-Val-Phe-Phe; SEQ ID NO: 10).

[0138] In another specific embodiment, the compound comprises theformula: A-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-B (e.g., a modified form ofAβ₁₆₋₂₁, comprising an amino acid sequence Lys-Leu-Val-Phe-Phe-Ala; SEQID NO: 9).

[0139] In another specific embodiment, the compound comprises theformula: A-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-B (e.g., a modified form ofAP₁₅₋₂₀, comprising an amino acid sequence Gln-Lys-Leu-Val-Phe-Phe; SEQID NO: 8).

[0140] In another specific embodiment, the compound comprises theformula: A-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-B (e.g., a modified formof AP₁₅₋₂₁, comprising an amino acid sequenceGln-Lys-Leu-Val-Phe-Phe-Ala; SEQ ID NO: 7).

[0141] In another specific embodiment, the compound comprises theformula: A-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-B (e.g., a modified formof AP 14-20, comprising an amino acid sequenceHis-Gln-Lys-Leu-Val-Phe-Phe; SEQ ID NO: 6).

[0142] In another specific embodiment, the compound comprises theformula: A-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-B (e.g., a modifiedform of AP₁₄₋₂₁, comprising an amino acid sequenceHis-Gln-Lys-Leu-Val-Phe-Phe-Ala; SEQ ID NO: 5).

[0143] In preferred embodiments of the aforementioned specificembodiments, A or B is a cholanoyl structure or a biotin-containingstructure (described further in subsection II below).

[0144] In further experiments to delineate subregions of Aβ upon whichan Aβ aggregation core domain can be modeled (the results of which aredescribed in Example 11), it was demonstrated that a modulator compoundhaving inhibitory activity can comprise as few as three AP amino acidsresidues (e.g., Val-Phe-Phe, which corresponds to Aβ₁₈₋₂₀ orPhe-Phe-Ala, which corresponds to Aβ₁₉₋₂₁). The results alsodemonstrated that a modulator compound having a modulating group at itscarboxy-terminus is effective at inhibiting Aβ aggregation. Stillfurther, the results demonstrated that the cholyl group, as a modulatinggroup, can be manipulated while maintaining the inhibitory activity ofthe compounds and that an iodotyrosyl can be substituted forphenylalanine (e.g., at position 19 or 20 of the Aβ sequence) whilemaintaining the ability of the compound to inhibit Aβ aggregation.

[0145] Still further, the results demonstrated that compounds withinhibitory activity can be created using amino acids residues that arederived from the Aβ sequence in the region of about positions 17-21 butwherein the amino acid sequence is rearranged or has a substitution witha non-Aβ-derived amino acid. Examples of such compounds include PPI-426,in which the sequence of AP₁₇₋₂₁ (LVFFA) has been rearranged (FFVLA),PPI-372, in which the sequence of A≢₁₆₋₂₀ (KLVFF) has been rearranged(FKFVL), and PPI-388, -389 and -390, in which the sequence of Aβ₁₇₋₂₁(LVFFA) has been substituted at position 17, 18 or 19, respectively,with an alanine residue (AVFFA for PPI-388, LAFFA for PPI-389 and LVAFAfor PPI-390). The inhibitory activity of these compounds indicate thatthe presence in the compound of an amino acid sequence directlycorresponding to a portion of Aβ is not essential for inhibitoryactivity, but rather suggests that maintenance of the hydrophobic natureof this core region, by inclusion of amino acid residues such asphenylalanine, valine, leucine, regardless of their precise order, canbe sufficient for inhibition of Aβ aggregation. Accordingly, an Aβaggregation core domain can be designed based on the direct Aβ aminoacid sequence or can be designed based on a rearranged AP sequence whichmaintains the hydrophobicity of the Aβ subregion, e.g., the regionaround positions 17-20. This region of Aβ contains the amino acidresidues Leu, Val and Phe. Accordingly, preferred AP aggregation coredomains are composed of at least three amino acid structures (as thatterm is defined hereinbefore, including amino acid derivatives,analogues and mimetics), wherein-at least two of the amino acidstructures are, independently, either a leucine structure, a valinestructure or a phenylalanine structure (as those terms are definedhereinbefore, including derivatives, analogues and mimetics).

[0146] Thus, in another embodiment, the invention provides a β-amyloidmodulator compound comprising a formula:

[0147] wherein

[0148] Xaa₁, Xaa₂ and Xaa₃ are each amino acid structures and at leasttwo of Xaa₁, Xaa₂ and Xaa₃ are, independently, selected from the groupconsisting of a leucine structure, a phenylalanine structure and avaline structure;

[0149] Y, which may or may not be present, is a peptidic structurehaving the formula (Xaa)_(a), wherein Xaa is any amino acid structureand a is an integer from 1 to 15;

[0150] Z, which may or may not be present, is a peptidic structurehaving the formula (Xaa)_(b), wherein Xaa is any amino acid structureand b is an integer from 1 to 15; and

[0151] A is a modifying group attached directly or indirectly to thecompound and n is an integer;

[0152] Xaa₁, Xaa₂, Xaa₃, Y, Z, A and n being selected such that thecompound modulates the aggregation or inhibits the neurotoxicity ofnatural β-amyloid peptides when contacted with the natural β-amyloidpeptides.

[0153] Preferably, the compound inhibits aggregation of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides.In preferred embodiments, Xaa₁ and Xaa₂ are each phenylalaninestructures or Xaa₂ and Xaa₃ are each phenylalanine structures. “n” canbe, for example, an integer between 1 and 5, whereas “a” and “b” can be,for example, integers between 1 and 5. The modifying group “A”preferably comprises a cyclic, heterocyclic or polycyclic group. Morepreferably, A contains a cis-decalin group, such as cholanoyl structureor a cholyl group In other embodiments, A can comprise abiotin-containing group, a diethylene-triaminepentaacetyl group, a(−)-menthoxyacetyl group, a fluorescein-containing group or anN-acetylneuraminyl group. In yet other embodiments, the compound maypromotes aggregation of natural β-amyloid peptides when contacted withthe natural β-amyloid peptides, may be further modified to alter apharmacokinetic property of the compound or may be further modified tolabel the compound with a detectable substance.

[0154] In another embodiment, the invention provides a β-amyloidmodulator compound comprising a formula:

A-(Y)-Xaa₁-Xaa₂-Xaa₃-(Z)-B

[0155] wherein

[0156] Xaa₁, Xaa₂ and Xaa₃ are each amino acid structures and at leasttwo of Xaa₁, Xaa₂ and Xaa₃ are, independently, selected from the groupconsisting of a leucine structure, a phenylalanine structure and avaline structure;

[0157] Y, which may or may not be present, is a peptidic structurehaving the formula (Xaa)_(a), wherein Xaa is any amino acid structureand a is an integer from 1 to 15;

[0158] Z, which may or may not be present, is a peptidic structurehaving the formula (Xaa)_(b), wherein Xaa is any amino acid structureand b is an integer from 1 to 15; and

[0159] A and B, at least one of which is present, are modifying groupsattached directly or indirectly to the amino terminus and carboxyterminus, respectively, of the compound;

[0160] Xaa₁, Xaa₂, Xaa₃, Y, Z, A and B being selected such that thecompound modulates the aggregation or inhibits the neurotoxicity ofnatural β-amyloid peptides when contacted with the natural β-amyloidpeptides.

[0161] Preferably, the compound inhibits aggregation of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides.In preferred embodiments, Xaa₁ and Xaa₂ are each phenylalaninestructures or Xaa₂ and Xaa₃ are each phenylalanine structures. In onesubembodiment, the compound comprises the formula:

A-(Y)-Xaa₁-Xaa₂-Xaa₃-(Z)

[0162] In another subembodiment, the compound comprises the formula:

(Y)-Xaa₁-Xaa₂-Xaa₃-(Z)-B

[0163] “n” can be, for example, an integer between 1 and 5, whereas “a”and “b” can be, for example, integers between 1 and 5. The modifyinggroup “A” preferably comprises a cyclic, heterocyclic or polycyclicgroup. More preferably, A contains a cis-decalin group, such ascholanoyl structure or a cholyl group In other embodiments, A cancomprise a biotin-containing group, a diethylene-triaminepentaacetylgroup, a (−)-menthoxyacetyl group, a fluorescein-containing group or anN-acetylneuraminyl group. In yet other embodiments, the compound maypromote aggregation of natural β-amyloid peptides when contacted withthe natural β-amyloid peptides, may be further modified to alter apharmacokinetic property of the compound or may be further modified tolabel the compound with a detectable substance.

[0164] In preferred specific embodiments, the invention provides aβ-amyloid modulator compound comprising a modifying group attacheddirectly or indirectly to a peptidic structure, wherein the peptidicstructure comprises amino acid structures having an amino acid sequenceselected from the group consisting of His-Gln-Lys-Leu-Val-Phe-Phe-Ala(SEQ ID NO: 5), His-Gln-Lys-Leu-Val-Phe-Phe (SEQ ID NO: 6),Gln-Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: 7), Gln-Lys-Leu-Val-Phe-Phe (SEQID NO: 8), Lys-Leu-Val-Phe-Phe-Ala (SEQ ID NO: 9), Lys-Leu-Val-Phe-Phe(SEQ ID NO: 10), Leu-Val-Phe-Phe-Ala (SEQ ID NO: 11), Leu-Val-Phe-Phe(SEQ ID NO: 12), Leu-Ala-Phe-Phe-Ala (SEQ ID NO: 13), Val-Phe-Phe (SEQID NO: 19), Phe-Phe-Ala (SEQ ID NO: 20), Phe-Phe-Val-Leu-Ala (SEQ ID NO:21), Leu-Val-Phe-Phe-Lys (SEQ ID NO: 22), Leu-Val-Iodotyrosine-Phe-Ala(SEQ ID NO: 23), Val-Phe-Phe-Ala (SEQ ID NO: 24), Ala-Val-Phe-Phe-Ala(SEQ ID NO: 25), Leu-Val-Phe-Iodotyrosine-Ala (SEQ ID NO: 26),Leu-Val-Phe-Phe-Ala-Glu (SEQ ID NO: 27), Phe-Phe-Val-Leu (SEQ ID NO:28), Phe-Lys-Phe-Val-Leu (SEQ ID NO: 29), Lys-Leu-Val-Ala-Phe (SEQ IDNO: 30), Lys-Leu-Val-Phe-Phe-βAla (SEQ ID NO: 31) andLeu-Val-Phe-Phe-DAla (SEQ ID NO: 32).

[0165] These specific compounds can be further modified to alter apharmacokinetic property of the compound and/or further modified tolabel the compound with a detectable substance.

[0166] The modulator compounds of the invention can be incorporated intopharmaceutical compositions (described further in subsection V below)and can be used in detection and treatment methods as described furtherin subsection VI below.

[0167] II. Modifying Groups

[0168] Within a modulator compound of the invention, a peptidicstructure (such as an Aβ derived peptide, or an Aβ aggregation coredomain, or an amino acid sequence corresponding to a rearranged Aβaggregation core domain) is coupled directly or indirectly to at leastone modifying group (abbreviated as MG). In one embodiment, a modulatorcompounds of the invention comprising an aggregation core domain coupledto a modifying group, the compound can be illustrated schematically asMG-ACD. The term “modifying group” is intended to include structuresthat are directly attached to the peptidic structure (e.g., by covalentcoupling), as well as those that are indirectly attached to the peptidicstructure (e.g., by a stable non-covalent association or by covalentcoupling to additional amino acid residues, or mimetics, analogues orderivatives thereof, which may flank the AP-derived peptidic structure).For example, the modifying group can be coupled to the amino-terminus orcarboxy-terminus of an Aβ-derived peptidic structure, or to a peptidicor peptidomimetic region flanking the core domain. Alternatively, themodifying group can be coupled to a side chain of at least one aminoacid residue of an Aβ-derived peptidic structure, or to a peptidic orpeptidomimetic region flanking the core domain (e.g., through theepsilon amino group of a lysyl residue(s), through the carboxyl group ofan aspartic acid residue(s) or a glutamic acid residue(s), through ahydroxy group of a tyrosyl residue(s), a serine residue(s) or athreonine residue(s) or other suitable reactive group on an amino acidside chain). Modifying groups covalently coupled to the peptidicstructure can be attached by means and using methods well known in theart for linking chemical structures, including, for example, amide,alkylamino, carbamate or urea bonds.

[0169] The term “modifying group” is intended to include groups that arenot naturally coupled to natural Aβ peptides in their native form.Accordingly, the term “modifying group” is not intended to includehydrogen. The modifying group(s) is selected such that the modulatorcompound alters, and preferably inhibits, aggregation of naturalβ-amyloid peptides when contacted with the natural β-amyloid peptides orinhibits the neurotoxicity of natural β-amyloid peptides when contactedwith the natural β-amyloid peptides. Although not intending to belimited by mechanism, the modifying group(s) of the modulator compoundsof the invention is thought to function as a key pharmacophore which isimportant for conferring on the modulator the ability to disrupt Aβpolymerization.

[0170] In a preferred embodiment, the modifying group(s) comprises acyclic, heterocyclic or polycyclic group. The term “cyclic group”, asused herein, is intended to include cyclic saturated or unsaturated(i.e., aromatic) group having from about 3 to 10, preferably about 4 to8, and more preferably about 5 to 7, carbon atoms. Exemplary cyclicgroups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, andcyclooctyl. Cyclic groups may be unsubstituted or substituted at one ormore ring positions. Thus, a cyclic group may be substituted with, e.g.,halogens, alkyls, cycloalkyls, alkenyls, alkynyls, aryls, heterocycles,hydroxyls, aminos, nitros, thiols amines, imines, amides, phosphonates,phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls,sulfonates, selenoethers, ketones, aldehydes, esters, —CF₃, —CN, or thelike.

[0171] The term “heterocyclic group” is intended to include cyclicsaturated or unsaturated (i.e., aromatic) group having from about 3 to10, preferably about 4 to 8, and more preferably about 5 to 7, carbonatoms, wherein the ring structure includes about one to fourheteroatoms. Heterocyclic groups include pyrrolidine, oxolane, thiolane,imidazole, oxazole, piperidine, piperazine, morpholine. The heterocyclicring can be substituted at one or more positions with such substituentsas, for example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls,aryls, other heterocycles, hydroxyl, amino, nitro, thiol, amines,imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls,ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters,—CF₃, —CN, or the like. Heterocycles may also be bridged or fused toother cyclic groups as described below.

[0172] The term “polycyclic group” as used herein is intended to referto two or more saturated or unsaturated (i.e., aromatic) cyclic rings inwhich two or more carbons are common to two adjoining rings, e.g., therings are “fused rings”. Rings that are joined through non-adjacentatoms are termed “bridged” rings. Each of the rings of the polycyclicgroup can be substituted with such substituents as described above, asfor example, halogens, alkyls, cycloalkyls, alkenyls, alkynyls,hydroxyl, amino, nitro, thiol, amines, imines, amides, phosphonates,phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls,selenoethers, ketones, aldehydes, esters, —CF₃, —CN, or the like.

[0173] A preferred polycyclic group is a group containing a cis-decalinstructure. Although not intending to be limited by mechanism, it isthought that the “bent” conformation conferred on a modifying group bythe presence of a cis-decalin structure contributes to the efficacy ofthe modifying group in disrupting AP polymerization. Accordingly, otherstructures which mimic the “bent” configuration of the cis-decalinstructure can also be used as modifying groups. An example of acis-decalin containing structure that can be used as a modifying groupis a cholanoyl structure, such as a cholyl group. For example, amodulator compound can be modified at its amino terminus with a cholylgroup by reacting the aggregation core domain with cholic acid, a bileacid, as described in Example 4 (the structure of cholic acid isillustrated in FIG. 2). Moreover, a modulator compound can be modifiedat its carboxy terminus with a cholyl group according to methods knownin the art (see e.g., Wess, G. et al. (1993) Tetrahedron Letters,34:817-822; Wess, G. et al. (1992) Tetrahedron Letters 33:195-198; andKramer, W. et al. (1992) J. Biol. Chem. 267:18598-18604). Cholylderivatives and analogues can also be used as modifying groups. Forexample, a preferred cholyl derivative is Aic(3-(O-aminoethyl-iso)-cholyl), which has a free amino group that can beused to further modify the modulator compound (e.g., a chelation groupfor ^(99m)Tc can be introduced through the free amino group of Aic). Asused herein, the term “cholanoyl structure” is intended to include thecholyl group and derivatives and analogues thereof, in particular thosewhich retain a four-ring cis-decalin configuration. Examples ofcholanoyl structures include groups derived from other bile acids, suchas deoxycholic acid, lithocholic acid, ursodeoxycholic acid,chenodeoxycholic acid and hyodeoxycholic acid, as well as other relatedstructures such as cholanic acid, bufalin and resibufogenin (althoughthe latter two compounds are not preferred for use as a modifyinggroup). Another example of a cis-decalin containing compound is5β-cholestan-3α-ol (the cis-decalin isomer of (+)-dihydrocholesterol).For further description of bile acid and steroid structure andnomenclature, see Nes, W. R. and McKean, M. L. Biochemistry of Steroidsand Other Isopentanoids, University Park Press, Baltimore, Md., Chapter2.

[0174] In addition to cis-decalin containing groups, other polycyclicgroups may be used as modifying groups. For example, modifying groupsderived from steroids or β-lactams may be suitable modifying groups.Moreover, non-limiting examples of some additional cyclic, heterocyclicor polycyclic compounds which can be used to modify an AP-derivedpeptidic structure are shown schematically in FIG. 2. In one embodiment,the modifying group is a “biotinyl structure”, which includes biotinylgroups and analogues and derivatives thereof (such as a 2-iminobiotinylgroup). In another embodiment, the modifying group can comprise a“fluorescein-containing group”, such as a group derived from reacting anAβ-derived peptidic structure with 5-(and 6-)-carboxyfluorescein,succinimidyl ester or fluorescein isothiocyanate. In various otherembodiments, the modifying group(s) can comprise an N-acetylneuraminylgroup, a trans-4-cotininecarboxyl group, a 2-imino-1-imidazolidineacetylgroup, an (S)-(−)-indoline-2-carboxyl group, a (−)-menthoxyacetyl group,a 2-norbornaneacetyl group, a γ-oxo-5-acenaphthenebutyryl, a(−)-2-oxo-4-thiazolidinecarboxyl group, a tetrahydro-3-furoyl group, a2-iminobiotinyl group, a diethylenetriaminepentaacetyl group, a4-morpholinecarbonyl group, a 2-thiopheneacetyl group or a2-thiophenesulfonyl group.

[0175] Preferred modifying groups include groups comprising cholylstructures, biotinyl structures, fluorescein-containing groups, adiethylene-triaminepentaacetyl group, a (−)-menthoxyacetyl group, and aN-acetylneuraminyl group. More preferred modifying groups thosecomprising a cholyl structure or an iminiobiotinyl group.

[0176] In addition to the cyclic, heterocyclic and polycyclic groupsdiscussed above, other types of modifying groups can be used in amodulator of the invention. For example, small hydrophobic groups may besuitable modifying groups. An example of a suitable non-cyclic modifyinggroup is an acetyl group.

[0177] Yet another type of modifying group is a compound that contains anon-natural amino acid that acts as a beta-turn mimetic, such as adibenzofuran-based amino acid described in Tsang, K. Y. et al. (1994) J.Am. Chem. Soc. 116:3988-4005; Diaz, H and Kelly, J. W. (1991)Tetrahedron Letters 41:5725-5728; and Diaz. H et al. (1992) J. Am. Chem.Soc. 114:8316-8318. An example of such a modifying group is apeptide-aminoethyldibenzofuranyl-proprionic acid (Adp) group (e.g.,DDIIL-Adp). This type of modifying group further can comprise one ormore N-methyl peptide bonds to introduce additional steric hindrance tothe aggregation of natural β-AP when compounds of this type interactwith natural β-AP.

[0178] III. Additional Chemical Modifications of Aβ Modulators

[0179] A β-amyloid modulator compound of the invention can be furthermodified to alter the specific properties of the compound whileretaining the ability of the compound to alter Aβ aggregation andinhibit Aβ neurotoxicity. For example, in one embodiment, the compoundis further modified to alter a pharmacokinetic property of the compound,such as in vivo stability or half-life. In another embodiment, thecompound is further modified to label the compound with a detectablesubstance. In yet another embodiment, the compound is further modifiedto couple the compound to an additional therapeutic moiety.Schematically, a modulator of the invention comprising an Aβ aggregationcore domain coupled directly or indirectly to at least one modifyinggroup can be illustrated as MG-ACD, whereas this compound which has beenfurther modified to alter the properties of the modulator can beillustrated as MG-ACD-CM, wherein CM represents an additional chemicalmodification.

[0180] To further chemically modify the compound, such as to alter thepharmacokinetic properties of the compound, reactive groups can bederivatized. For example, when the modifying group is attached to theamino-terminal end of the aggregation core domain, the carboxy-terminalend of the compound can be further modified. Preferred C-terminalmodifications include those which reduce the ability of the compound toact as a substrate for carboxypeptidases. Examples of preferredC-terminal modifiers include an amide group, an ethylamide group andvarious non-natural amino acids, such as D-amino acids and β-alanine.Alternatively, when the modifying group is attached to thecarboxy-terminal end of the aggregation core domain, the amino-terminalend of the compound can be further modified, for example, to reduce theability of the compound to act as a substrate for aminopeptidases.

[0181] A modulator compound can be further modified to label thecompound by reacting the compound with a detectable substance. Suitabledetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials and radioactive materials.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ¹⁴C, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I,^(99m)Tc, ³⁵S or ³H. In a preferred embodiment, a modulator compound isradioactively labeled with ¹⁴C, either by incorporation of ¹⁴C into themodifying group or one or more amino acid structures in the modulatorcompound. Labeled modulator compounds can be used to assess the in vivopharmacokinetics of the compounds, as well as to detect Aβ aggregation,for example for diagnostic purposes. Aβ aggregation can be detectedusing a labeled modulator compound either in vivo or in an in vitrosample derived from a subject.

[0182] Preferably, for use as an in vivo diagnostic agent, a modulatorcompound of the invention is labeled with radioactive technetium oriodine. Accordingly, in one embodiment, the invention provides amodulator compound labeled with technetium, preferably ^(99m)Tc. Methodsfor labeling peptide compounds with technetium are known in the art (seee.g., U.S. Pat. Nos. 5,443,815, 5,225,180 and 5,405,597, all by Dean etal.; Stepniak-Biniakiewicz, D., et al. (1992) J. Med. Chem. 35:274-279;Fritzberg, A. R., et al. (1988) Proc. Natl. Acad. Sci. USA 85:4025-4029;Baidoo, K. E., et al. (1990) Cancer Res. Suppl. 50:799s-803s; and Regan,L. and Smith, C. K. (1995) Science 270:980-982). A modifying group canbe chosen that provides a site at which a chelation group for ^(99m)Tccan be introduced, such as the Aic derivative of cholic acid, which hasa free amino group (see Example 11). In another embodiment, theinvention provides a modulator compound labeled with radioactive iodine.For example, a phenylalanine residue within the Aβ sequence (such asPhe₁₉ or Phe₂₀) can be substituted with radioactive iodotyrosyl (seeExample 11). Any of the various isotopes of radioactive iodine can beincorporated to create a diagnostic agent. Preferably, ¹²³I(half-life=13.2 hours) is used for whole body scintigraphy, ¹²⁴I (halflife=4 days) is used for positron emission tomography (PET), ¹²⁵I (halflife=60 days) is used for metabolic turnover studies and ¹³¹I (halflife=8 days) is used for whole body counting and delayed low resolutionimaging studies.

[0183] Furthermore, an additional modification of a modulator compoundof the invention can serve to confer an additional therapeutic propertyon the compound. That is, the additional chemical modification cancomprise an additional functional moiety. For example, a functionalmoiety which serves to break down or dissolve amyloid plaques can becoupled to the modulator compound. In this form, the MG-ACD portion ofthe modulator serves to target the compound to Aβ peptides and disruptthe polymerization of the Aβ peptides, whereas the additional functionalmoiety serves to break down or dissolve amyloid plaques after thecompound has been targeted to these sites.

[0184] In an alternative chemical modification, a β-amyloid compound ofthe invention is prepared in a “prodrug” form, wherein the compounditself does not modulate Aβ aggregation, but rather is capable of beingtransformed, upon metabolism in vivo, into a β-amyloid modulatorcompound as defined herein. For example, in this type of compound, themodulating group can be present in a prodrug form that is capable ofbeing converted upon metabolism into the form of an active modulatinggroup. Such a prodrug form of a modifying group is referred to herein asa “secondary modifying group.” A variety of strategies are known in theart for preparing peptide prodrugs that limit metabolism in order tooptimize delivery of the active form of the peptide-based drug (seee.g., Moss, J. (1995) in Peptide-Based Drug Design: ControllingTransport and Metabolism, Taylor, M. D. and Amidon, G. L. (eds), Chapter18. Additionally strategies have been specifically tailored to achievingCNS delivery based on “sequential metabolism” (see e.g., Bodor, N., etal. (1992) Science 257:1698-1700; Prokai, L., et al. (1994) J. Am. Chem.Soc. 116:2643-2644; Bodor, N. and Prokai, L. (1995) in Peptide-BasedDrug Design: Controlling Transport and Metabolism, Taylor, M. D. andAmidon, G. L. (eds), Chapter 14. In one embodiment of a prodrug form ofa modulator of the invention, the modifying group comprises an alkylester to facilitate blood-brain barrier permeability.

[0185] Modulator compounds of the invention can be prepared by standardtechniques known in the art. The peptide component of a modulatorcomposed, at least in part, of a peptide, can be synthesized usingstandard techniques such as those described in Bodansky, M. Principlesof Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant, G. A(ed.). Synthetic Peptides: A User's Guide, W. H. Freeman and Company,New York (1992). Automated peptide synthesizers are commerciallyavailable (e.g., Advanced ChemTech Model 396; Milligen/Biosearch 9600).Additionally, one or more modulating groups can be attached to theAβ-derived peptidic component (e.g., an Aβ aggregation core domain) bystandard methods, for example using methods for reaction through anamino group (e.g., the alpha-amino group at the amino-terminus of apeptide), a carboxyl group (e.g., at the carboxy terminus of a peptide),a hydroxyl group (e.g., on a tyrosine, serine or threonine residue) orother suitable reactive group on an amino acid side chain (see e.g.,Greene, T. W and Wuts, P. G. M. Protective Groups in Organic Synthesis,John Wiley and Sons, Inc., New York (1991). Exemplary syntheses ofpreferred β amyloid modulators is described further in Examples 1, 4 and11.

[0186] IV. Screening Assays

[0187] Another aspect of the invention pertains to a method forselecting a modulator of β-amyloidaggregation. In the method, a testcompound is contacted with natural β amyloid peptides, the aggregationof the natural β-AP is measured and a modulator is selected based on theability of the test compound to alter the aggregation of the naturalβ-AP (e.g., inhibit or promote aggregation). In a preferred embodiment,the test compound is contacted with a molar excess amount of the naturalβ-AP. The amount and/or rate of natural β-AP aggregation in the presenceof the test compound can be determined by a suitable assay indicative ofβ-AP aggregation, as described herein (see e.g., Examples 2, 5 and 6).

[0188] In a preferred assay, the natural β-AP is dissolved in solutionin the presence of the test compound and aggregation of the natural β-APis assessed in a nucleation assay (see Example 6) by assessing theturbidity of the solution over time, as measured by the apparentabsorbance of the solution at 405 nm (described further in Example 6;see also Jarrett et al. (1993) Biochemistry 32:4693-4697). In theabsence of a β-amyloid modulator, the A_(405 nm) of the solutiontypically stays relatively constant during a lag time in which the β-APremains in solution, but then the A_(405 nm) of the solution rapidlyincreases as the β-AP aggregates and comes out of solution, ultimatelyreaching a plateau level (i.e., the A_(405 nm) of the solution exhibitssigmoidal kinetics over time). In contrast, in the presence of a testcompound that inhibits β-AP aggregation, the A_(405 nm) of the solutionis reduced compared to when the modulator is absent. Thus, in thepresence of the inhibitory modulator, the solution may exhibit anincreased lag time, a decreased slope of aggregation and/or a lowerplateau level compared to when the modulator is absent. This method forselecting a modulator of β-amyloid polymerization can similarly be usedto select modulators that promote β-AP aggregation. Thus, in thepresence of a modulator that promotes β-AP aggregation, the A_(405 nm)of the solution is increased compared to when the modulator is absent(e.g., the solution may exhibit an decreased lag time, increase slope ofaggregation and/or a higher plateau level compared to when the modulatoris absent).

[0189] Another assay suitable for use in the screening method of theinvention, a seeded extension assay, is also described further inExample 6. In this assay, β-AP monomer and an aggregated β-AP “seed” arecombined, in the presence and absence of a test compound, and the amountof β-fibril formation is assayed based on enhanced emission of the dyeThioflavine T when contacted with β-AP fibrils. Moreover, β-APaggregation can be assessed by electron microscopy (EM) of the β-APpreparation in the presence or absence of the modulator. For example, βamyloid fibril formation, which is detectable by EM, is reduced in thepresence of a modulator that inhibits β-AP aggregation (i.e., there is areduced amount or number of β-fibrils in the presence of the modulator),whereas β fibril formation is increased in the presence of a modulatorthat promotes β-AP aggregation (i.e., there is an increased amount ornumber of β-fibrils in the presence of the modulator).

[0190] An even more preferred assay for use in the screening method ofthe invention to select suitable modulators is the neurotoxicity assaydescribed in Examples 3 and 10. Compounds are selected which inhibit theformation of neurotoxic Aβ aggregates and/or which inhibit theneurotoxicity of preformed Aβ fibrils. This neurotoxicity assay isconsidered to be predictive of neurotoxicity in vivo. Accordingly,inhibitory activity of a modulator compound in the in vitroneurotoxicity assay is predictive of similar inhibitory activity of thecompound for neurotoxicity in vivo.

[0191] V. Pharmaceutical Compositions

[0192] Another aspect of the invention pertains to pharmaceuticalcompositions of the β-amyloid modulator compounds of the invention. Inone embodiment, the composition includes a β amyloid modulator compoundin a therapeutically or prophylactically effective amount sufficient toalter, and preferably inhibit, aggregation of natural β-amyloidpeptides, and a pharmaceutically acceptable carrier. In anotherembodiment, the composition includes a β amyloid modulator compound in atherapeutically or prophylactically effective amount sufficient toinhibit the neurotoxicity of natural β-amyloid peptides, and apharmaceutically acceptable carrier. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired therapeutic result, such asreduction or reversal or β-amyloid deposition and/or reduction orreversal of Aβ neurotoxicity. A therapeutically effective amount ofmodulator may vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of the modulator toelicit a desired response in the individual. Dosage regimens may beadjusted to provide the optimum therapeutic response. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the modulator are outweighed by the therapeutically beneficialeffects. The potential neurotoxicity of the modulators of the inventioncan be assayed using the cell-based assay described in Examples 3 and 10and a therapeutically effective modulator can be selected which does notexhibit significant neurotoxicity. In a preferred embodiment, atherapeutically effective amount of a modulator is sufficient to alter,and preferably inhibit, aggregation of a molar excess amount of naturalβ-amyloid peptides. A “prophylactically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired prophylactic result, such as preventing orinhibiting the rate of β-amyloid deposition and/or Aβ neurotoxicity in asubject predisposed to β-amyloid deposition. A prophylacticallyeffective amount can be determined as described above for thetherapeutically effective amount. Typically, since a prophylactic doseis used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

[0193] One factor that may be considered when determining atherapeutically or prophylactically effective amount of a β amyloidmodulator is the concentration of natural β-AP in a biologicalcompartment of a subject, such as in the cerebrospinal fluid (CSF) ofthe subject. The concentration of natural β-AP in the CSF has beenestimated at 3 nM (Schwartzman, (1994) Proc. Natl. Acad. Sci. USA91:8368-8372). A non-limiting range for a therapeutically orprophylactically effective amounts of a β amyloid modulator is 0.01nM-10 μM. It is to be noted that dosage values may vary with theseverity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions, and that dosage ranges set forthherein are exemplary only and are not intended to limit the scope orpractice of the claimed composition.

[0194] The amount of active compound in the composition may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, each of which may affect the amount of natural β-AP inthe individual. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

[0195] As used herein “pharmaceutically acceptable carrier” includes anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and the likethat are physiologically compatible. In one embodiment, the carrier issuitable for parenteral administration. Preferably, the carrier issuitable for administration into the central nervous system (e.g.,intraspinally or intracerebrally). Alternatively, the carrier can besuitable for intravenous, intraperitoneal or intramuscularadministration. In another embodiment, the carrier is suitable for oraladministration. Pharmaceutically acceptable carriers include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active compound, use thereof inthe pharmaceutical compositions of the invention is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

[0196] Therapeutic compositions typically must be sterile and stableunder the conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, liposome, or other orderedstructure suitable to high drug concentration. The carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. In many cases, itwill be preferable to include isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, or sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, monostearate salts and gelatin. Moreover, themodulators can be administered in a time release formulation, forexample in a composition which includes a slow release polymer. Theactive compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers(PLG). Many methods for the preparation of such formulations arepatented or generally known to those skilled in the art.

[0197] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., β-amyloid modulator) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.

[0198] A modulator compound of the invention can be formulated with oneor more additional compounds that enhance the solubility of themodulator compound. Preferred compounds to be added to formulations toenhance the solubility of the modulators are cyclodextrin derivatives,preferably hydroxypropyl-γ-cyclodextrin. Drug delivery vehiclescontaining a cyclodextrin derivative for delivery of peptides to thecentral nervous system are described in Bodor, N., et al. (1992) Science257:1698-1700. For the β-amyloid modulators described herein, inclusionin the formulation of hydroxypropyl-γ-cyclodextrin at a concentration50-200 mM increases the aqueous solubility of the compounds. In additionto increased solubility, inclusion of a cyclodextrin derivative in theformulation may have other beneficial effects, since β-cyclodextrinitself has been reported to interact with the AP peptide and inhibitfibril formation in vitro (Camilleri, P., et al (1994) FEBS Letters341:256-258. Accordingly, use of a modulator compound of the inventionin combination with a cyclodextrin derivative may result in greaterinhibition of Aβ aggregation than use of the modulator alone. Chemicalmodifications of cyclodextrins are known in the art (Hanessian, S., etal. (1995) J. Org. Chem. 60:4786-4797). In addition to use as anadditive in a pharmaceutical composition containing a modulator of theinvention, cyclodextrin derivatives may also be useful as modifyinggroups and, accordingly, may also be covalently coupled to an Aβ peptidecompound to form a modulator compound of the invention.

[0199] In another embodiment, a pharmaceutical composition comprising amodulator of the invention is formulated such that the modulator istransported across the blood-brain barrier (BBB). Various strategiesknown in the art for increasing transport across the BBB can be adaptedto the modulators of the invention to thereby enhance transport of themodulators across the BBB (for reviews of such strategies, see e.g.,Pardridge, W. M. (1994) Trends in Biotechnol. 12:239-245; Van Bree, J.B. et al. (1993) Pharm. World Sci. 15:2-9; and Pardridge, W. M. et al.(1992) Pharmacol. Toxicol. 71:3-10). In one approach, the modulator ischemically modified to form a prodrug with enhanced transmembranetransport. Suitable chemical modifications include covalent linking of afatty acid to the modulator through an amide or ester linkage (see e.g.,U.S. Pat. No. 4,933,324 and PCT Publication WO 89/07938, both byShashoua; U.S. Pat. No. 5,284,876 by Hesse et al.; Toth, I. et al.(1994) J. Drug Target. 2:217-239; and Shashoua, V. E. et al. (1984) J.Med. Chem. 27:659-664) and glycating the modulator (see e.g., U.S. Pat.No. 5,260,308 by Poduslo et al.). Also, N-acylamino acid derivatives maybe used in a modulator to form a “lipidic” prodrug (see e.g., U.S. Pat.No. 5,112,863 by Hashimoto et al).

[0200] In another approach for enhancing transport across the BBB, apeptidic or peptidomimetic modulator is conjugated to a second peptideor protein, thereby forming a chimeric protein, wherein the secondpeptide or protein undergoes absorptive-mediated or receptor-mediatedtranscytosis through the BBB. Accordingly, by coupling the modulator tothis second peptide or protein, the chimeric protein is transportedacross the BBB. The second peptide or protein can be a ligand for abrain capillary endothelial cell receptor ligand. For example, apreferred ligand is a monoclonal antibody that specifically binds to thetransferrin receptor on brain capillary endothelial cells (see e.g.,U.S. Pat. Nos. 5,182,107 and 5,154,924 and PCT Publications WO 93/10819and WO 95/02421, all by Friden et al.). Other suitable peptides orproteins that can mediate transport across the BBB include histones (seee.g., U.S. Pat. No. 4,902,505 by Pardridge and Schimmel) and ligandssuch as biotin, folate, niacin, pantothenic acid, riboflavin, thiamin,pryridoxal and ascorbic acid (see e.g., U.S. Pat. Nos. 5,416,016 and5,108,921, both by Heinstein). Additionally, the glucose transporterGLUT-1 has been reported to transport glycopeptides(L-serinyl-β-D-glucoside analogues of [Met5]enkephalin) across the BBB(Polt, R. et al. (1994) Proc. Natl. Acad. Sci. USA 91:7114-1778).Accordingly, a modulator compound can be coupled to such a glycopeptideto target the modulator to the GLUT-1 glucose transporter. For example,a modulator compound which is modified at its amino terminus with themodifying group Aic (3-(O-aminoethyl-iso)-cholyl, a derivative of cholicacid having a free amino group) can be coupled to a glycopeptide throughthe amino group of Aic by standard methods. Chimeric proteins can beformed by recombinant DNA methods (e.g., by formation of a chimeric geneencoding a fusion protein) or by chemical crosslinking of the modulatorto the second peptide or protein to form a chimeric protein. Numerouschemical crosslinking agents are known in the (e.g., commerciallyavailable from Pierce, Rockford Ill.). A crosslinking agent can bechosen which allows for high yield coupling of the modulator to thesecond peptide or protein and for subsequent cleavage of the linker torelease bioactive modulator. For example, a biotin-avidin-based linkersystem may be used.

[0201] In yet another approach for enhancing transport across the BBB,the modulator is encapsulated in a carrier vector which mediatestransport across the BBB. For example, the modulator can be encapsulatedin a liposome, such as a positively charged unilamellar liposome (seee.g., PCT Publications WO 88/07851 and WO 88/07852, both by Faden) or inpolymeric microspheres (see e.g., U.S. Pat. No. 5,413,797 by Khan etal., U.S. Pat. No. 5,271,961 by Mathiowitz et al. and U.S. Pat. No.5,019,400 by Gombotz et al.). Moreover, the carrier vector can bemodified to target it for transport across the BBB. For example, thecarrier vector (e.g., liposome) can be covalently modified with amolecule which is actively transported across the BBB or with a ligandfor brain endothelial cell receptors, such as a monoclonal antibody thatspecifically binds to transferrin receptors (see e.g., PCT PublicationsWO 91/04014 by Collins et al and WO 94/02178 by Greig et al.).

[0202] In still another approach to enhancing transport of the modulatoracross the BBB, the modulator is coadministered with another agent whichfunctions to permeabilize the BBB. Examples of such BBB “permeabilizers”include bradykinin and bradykinin agonists (see e.g., U.S. Pat. No.5,112,596 by Malfroy-Camine) and peptidic compounds disclosed in U.S.Pat. No. 5,268,164 by Kozarich et al.

[0203] A modulator compound of the invention can be formulated into apharmaceutical composition wherein the modulator is the only activecompound or, alternatively, the pharmaceutical composition can containadditional active compounds. For example, two or more modulatorcompounds may be used in combination. Moreover, a modulator compound ofthe invention can be combined with one or more other agents that haveanti-amyloidogenic properties. For example, a modulator compound can becombined with the non-specific cholinesterase inhibitor tacrine (Cognex(W, Parke-Davis).

[0204] In another embodiment, a pharmaceutical composition of theinvention is provided as a packaged formulation. The packagedformulation may include a pharmaceutical composition of the invention ina container and printed instructions for administration of thecomposition for treating a subject having a disorder associated withβ-amyloidosis, e.g. Alzheimer's disease.

[0205] VI. Methods of Using Aβ Modulators

[0206] Another aspect of the invention pertains to methods for alteringthe aggregation or inhibiting the neurotoxicity of natural β-amyloidpeptides. In the methods of the invention, natural β amyloid peptidesare contacted with a β amyloid modulator such that the aggregation ofthe natural β amyloid peptides is altered or the neurotoxicity of thenatural β amyloid peptides is inhibited. In a preferred embodiment, themodulator inhibits aggregation of the natural β amyloid peptides. Inanother embodiment, the modulator promotes aggregation of the natural βamyloid peptides. Preferably, aggregation of a molar excess amount ofβ-AP, relative to the amount of modulator, is altered upon contact withthe modulator.

[0207] In the method of the invention, natural β amyloid peptides can becontacted with a modulator either in vitro or in vivo. Thus, the term“contacted with” is intended to encompass both incubation of a modulatorwith a natural β-AP preparation in vitro and delivery of the modulatorto a site in vivo where natural β-AP is present. Since the modulatorcompound interacts with natural β-AP, the modulator compounds can beused to detect natural β-AP, either in vitro or in vivo. Accordingly,one use of the modulator compounds of the invention is as diagnosticagents to detect the presence of natural β-AP, either in a biologicalsample or in vivo in a subject. Furthermore, detection of natural β-APutilizing a modulator compound of the invention further can be used todiagnose amyloidosis in a subject. Additionally, since the modulatorcompounds of the invention disrupt β-AP aggregation and inhibit β-APneurotoxicity, the modulator compounds also are useful in the treatmentof disorders associated with β-amyloidosis, either prophylactically ortherapeutically. Accordingly, another use of the modulator compounds ofthe invention is as therapeutic agents to alter aggregation and/orneurotoxicity of natural β-AP.

[0208] In one embodiment, a modulator compound of the invention is usedin vitro, for example to detect and quantitate natural β-AP in sample(e.g., a sample of biological fluid). To aid in detection, the modulatorcompound can be modified with a detectable substance. The source ofnatural β-AP used in the method can be, for example, a sample ofcerebrospinal fluid (e.g., from an AD patient, an adult susceptible toAD due to family history, or a normal adult). The natural β-AP sample iscontacted with a modulator of the invention and aggregation of the β-APis measured, such as by as assay described in Examples 2, 5 and 6.Preferably, the nucleation assay and/or seeded extension assay describedin Example 6 is used. The degree of aggregation of the S-AP sample canthen be compared to that of a control sample(s) of a known concentrationof β-AP, similarly contacted with the modulator and the results can beused as an indication of whether a subject is susceptible to or has adisorder associated with β-amyloidosis. Moreover, β-AP can be detectedby detecting a modulating group incorporated into the modulator. Forexample, modulators incorporating a biotin compound as described herein(e.g., an amino-terminally biotinylated β-AP peptide) can be detectedusing a streptavidin or avidin probe which is labeled with a detectablesubstance (e.g., an enzyme, such as peroxidase). Detection of naturalβ-AP aggregates mixed with a modulator of the invention using a probethat binds to the modulating group (e.g., biotin/streptavidin) isdescribed further in Example 2.

[0209] In another embodiment, a modulator compound of the invention isused in vivo to detect, and, if desired, quantitate, natural β-APdeposition in a subject, for example to aid in the diagnosis of βamyloidosis in the subject. To aid in detection, the modulator compoundcan be modified with a detectable substance, preferably ^(99m)Tc orradioactive iodine (described further above), which can be detected invivo in a subject. The labeled β-amyloid modulator compound isadministered to the subject and, after sufficient time to allowaccumulation of the modulator at sites of amyloid deposition, thelabeled modulator compound is detected by standard imaging techniques.The radioactive signal generated by the labeled compound can be directlydetected (e.g., whole body counting), or alternatively, the radioactivesignal can be converted into an image on an autoradiograph or on acomputer screen to allow for imaging of amyloid deposits in the subject.Methods for imaging amyloidosis using radiolabeled proteins are known inthe art. For example, serum amyloid P component (SAP), radiolabeled witheither ¹²³I or ^(99m)Tc, has been used to image systemic amyloidosis(see e.g., Hawkins, P. N. and Pepys, M. B. (1995) Eur. J. Nucl. Med.22:595-599). Of the various isotypes of radioactive iodine, preferably¹²³I (half-life=13.2 hours) is used for whole body scintigraphy, ¹²⁴I(half life=4 days) is used for positron emission tomography (PET), ¹²⁵I(half life=60 days) is used for metabolic turnover studies and ¹³¹I(half life=8 days) is used for whole body counting and delayed lowresolution imaging studies. Analogous to studies using radiolabeled SAP,a labeled modulator compound of the invention can be delivered to asubject by an appropriate route (e.g., intravenously, intraspinally,intracerebrally) in a single bolus, for example containing 100 μg oflabeled compound carrying approximately 180 MBq of radioactivity.

[0210] The invention provides a method for detecting the presence orabsence of natural β-amyloid peptides in a biological sample, comprisingcontacting a biological sample with a compound of the invention anddetecting the compound bound to natural β-amyloid peptides to therebydetect the presence or absence of natural β-amyloid peptides in thebiological sample. In one embodiment, the β-amyloid modulator compoundand the biological sample are contacted in vitro. In another embodiment,the β-amyloid modulator compound is contacted with the biological sampleby administering the β-amyloid modulator compound to a subject. For invivo administration, preferably the compound is labeled with radioactivetechnetium or radioactive iodine.

[0211] The invention also provides a method for detecting naturalβ-amyloid peptides to facilitate diagnosis of a β-amyloidogenic disease,comprising contacting a biological sample with the compound of theinvention and detecting the compound bound to natural β-amyloid peptidesto facilitate diagnosis of a β-amyloidogenic disease. In one embodiment,the β-amyloid modulator compound and the biological sample are contactedin vitro. In another embodiment, the β-amyloid modulator compound iscontacted with the biological sample by administering the β-amyloidmodulator compound to a subject. For in vivo administration, preferablythe compound is labeled with radioactive technetium or radioactiveiodine. Preferably, use of the method facilitates diagnosis ofAlzheimer's disease.

[0212] In another embodiment, the invention provides a method foraltering natural β-AP aggregation or inhibiting β-AP neurotoxicity,which can be used prophylactically or therapeutically in the treatmentor prevention of disorders associated with β amyloidosis, e.g.,Alzheimer's Disease. As demonstrated in Example 10, modulator compoundsof the invention reduce the toxicity of natural β-AP aggregates tocultured neuronal cells. Moreover, the modulators not only reduce theformation of neurotoxic aggregates but also have the ability to reducethe neurotoxicity of preformed Aβ fibrils. Accordingly, the modulatorcompounds of the invention can be used to inhibit or prevent theformation of neurotoxic Aβ fibrils in subjects (e.g., prophylacticallyin a subject predisposed to β-amyloid deposition) and can be used toreverse β-amyloidosis therapeutically in subjects already exhibitingβ-amyloid deposition.

[0213] A modulator of the invention is contacted with natural β amyloidpeptides present in a subject (e.g., in the cerebrospinal fluid orcerebrum of the subject) to thereby alter the aggregation of the naturalβ-AP and/or inhibit the neurotoxicity of the natural β-APs. A modulatorcompound alone can be administered to the subject, or alternatively, themodulator compound can be administered in combination with othertherapeutically active agents (e.g., as discussed above in subsectionIV). When combination therapy is employed, the therapeutic agents can becoadministered in a single pharmaceutical composition, coadministered inseparate pharmaceutical compositions or administered sequentially.

[0214] The modulator may be administered to a subject by any suitableroute effective for inhibiting natural β-AP aggregation in the subject,although in a particularly preferred embodiment, the modulator isadministered parenterally, most preferably to the central nervous systemof the subject. Possible routes of CNS administration includeintraspinal administration and intracerebral administration (e.g.,intracerebrovascular administration). Alternatively, the compound can beadministered, for example, orally, intraperitoneally, intravenously orintramuscularly. For non-CNS administration routes, the compound can beadministered in a formulation which allows for transport across the BBB.Certain modulators may be transported across the BBB without anyadditional further modification whereas others may need furthermodification as described above in subsection IV.

[0215] Suitable modes and devices for delivery of therapeutic compoundsto the CNS of a subject are known in the art, including cerebrovascularreservoirs (e.g., Ommaya or Rikker reservoirs; see e.g., Raney, J. P. etal. (1988) J. Neurosci. Nurs. 20:23-29; Sundaresan, N. et al. (1989)Oncology 3:15-22), catheters for intrathecal delivery (e.g.,Port-a-Cath, Y-catheters and the like; see e.g., Plummer, J. L. (1991)Pain 44:215-220; Yaksh, T. L. et al. (1986) Pharmacol. Biochem. Behav.25:483-485), injectable intrathecal reservoirs (e.g., Spinalgesic; seee.g., Brazenor, G. A. (1987) Neurosurgery 21:484-491), implantableinfusion pump systems (e.g., Infusaid; see e.g., Zierski, J. et al.(1988) Acta Neurochem. Suppl. 43:94-99; Kanoff, R. B. (1994) J. Am.Osteopath. Assoc. 94:487-493) and osmotic pumps (sold by AlzaCorporation). A particularly preferred mode of administration is via animplantable, externally programmable infusion pump. Suitable infusionpump systems and reservoir systems are also described in U.S. Pat. No.5, 368,562 by Blomquist and U.S. Pat. No. 4,731,058 by Doan, developedby Pharmacia Deltec Inc.

[0216] The method of the invention for altering β-AP aggregation invivo, and in particular for inhibiting β-AP aggregation, can be usedtherapeutically in diseases associated with abnormal β amyloidaggregation and deposition to thereby slow the rate of β amyloiddeposition and/or lessen the degree of β amyloid deposition, therebyameliorating the course of the disease. In a preferred embodiment, themethod is used to treat Alzheimer's disease (e.g., sporadic or familialAD, including both individuals exhibiting symptoms of AD and individualssusceptible to familial AD). The method can also be usedprophylactically or therapeutically to treat other clinical occurrencesof β amyloid deposition, such as in Down's syndrome individuals and inpatients with hereditary cerebral hemorrhage with amyloidosis-Dutch-type(HCHWA-D). While inhibition of β-AP aggregation is a preferredtherapeutic method, modulators that promote β-AP aggregation may also beuseful therapeutically by allowing for the sequestration of β-AP atsites that do not lead to neurological impairment.

[0217] Additionally, abnormal accumulation of β-amyloid precursorprotein in muscle fibers has been implicated in the pathology ofsporadic inclusion body myositis (IBM) (Askana, V. et al. (1996) Proc.Natl. Acad. Sci. USA 93:1314-1319; Askanas, V. et al. (1995) CurrentOpinion in Rheumatology 7:486-496). Accordingly, the modulators of theinvention can be used prophylactically or therapeutically in thetreatment of disorders in which β-AP, or APP, is abnormally deposited atnon-neurological locations, such as treatment of IBM by delivery of themodulators to muscle fibers.

[0218] VII. Unmodified Aβ Peptides that Inhibit Aggregation of Naturalβ-AP

[0219] In addition to the β-amyloid modulators described hereinbefore inwhich an Aβ peptide is coupled to a modifying group, the invention alsoprovides β-amyloid modulators comprised of an unmodified Aβ peptide. Ithas now been discovered that certain portions of natural β-AP can alteraggregation of natural β-APs when contacted with the natural β-APs (seeExample 12). Accordingly, these unmodified Aβ peptides comprise aportion of the natural β-AP sequence (i.e., a portion of βAP₁₋₃₉,βAP₁₋₄₀, βAP₁₋₄₂ and βAP₁₋₄₃). In particular these unmodified Aβpeptides have at least one amino acid deletion compared to βAP₁₋₃₉, theshortest natural β-AP, such that the compound alters aggregation ofnatural β-amyloid peptides when contacted with the natural β-amyloidpeptides. In various embodiments, these unmodified peptide compounds canpromote aggregation of natural β-amyloid peptides, or, more preferably,can inhibit aggregation of natural β-amyloid peptides when contactedwith the natural β-amyloid peptides. Even more preferably, theunmodified peptide compound inhibits aggregation of natural β-amyloidpeptides when contacted with a molar excess amount of natural β-amyloidpeptides (e.g., a 10-fold, 33-fold or 100-fold molar excess amount ofnatural β-AP).

[0220] As discussed above, the unmodified peptide compounds of theinvention comprise an amino acid sequence having at least one amino aciddeletion compared to the amino acid sequence of βAP₁₋₃₉. Alternatively,the unmodified peptide compound can have at least five, ten, fifteen,twenty, twenty-five, thirty or thirty-five amino acids deleted comparedto βAP₁₋₃₉. Still further the unmodified peptide compound can have1-5,1-10, 1-15, 1-20, 1-25, 1-30 or 1-35 amino acids deleted compared toβAP₁₋₃₉. The amino acid deletion(s) may occur at the amino-terminus, thecarboxy-terminus, an internal site, or a combination thereof, of theβ-AP sequence. Accordingly, in one embodiment, an unmodified peptidecompound of the invention comprises an amino acid sequence which has atleast one internal amino acid deleted compared to βAP₁₋₃₉.Alternatively, the unmodified peptide compound can have at least five,ten, fifteen, twenty, twenty-five, thirty or thirty-five internal aminoacids deleted compared to βAP₁₋₃₉. Still further the unmodified peptidecompound can have 1-5,1-10, 1-15, 1-20, 1-25, 1-30 or 1-35 internalamino acids deleted compared to βAP₁₋₃₉. For peptides with internaldeletions, preferably the peptide has an amino terminus corresponding toamino acid residue 1 of natural βAP and a carboxy terminus correspondingto residue 40 of natural βAP and has one or more internal β-AP aminoacid residues deleted (i.e., a non-contiguous Aβ peptide).

[0221] In another embodiment, the unmodified peptide compound comprisesan amino acid sequence which has at least one N-terminal amino aciddeleted compared to βAP₁₋₃₉ Alternatively, the unmodified peptidecompound can have at least five, ten, fifteen, twenty, twenty-five,thirty or thirty-five N-terminal amino acids deleted compared toβAP₁₋₃₉. Still further the unmodified peptide compound can have1-5,1-10, 1-15, 1-20, 1-25, 1-30 or 1-35 N-terminal amino acids deletedcompared to βAP₁₋₃₉.

[0222] In yet another embodiment, the unmodified peptide compoundcomprises an amino acid sequence which has at least one C-terminal aminoacid deleted compared to βAP₁₋₃₉. Alternatively, the unmodified peptidecompound can have at least five, ten, fifteen, twenty, twenty-five,thirty or thirty-five C-terminal amino acids deleted compared toβAP₁₋₃₉. Still further the unmodified peptide compound can have1-5,1-10, 1-15, 1-20, 1-25, 1-30 or 1-35 C-terminal amino acids deletedcompared to βAP₁₋₃₉.

[0223] In addition to deletion of amino acids as compared to βAP₁₋₃₉,the peptide compound can have additional non-β-AP amino acid residuesadded to it, for example, at the amino terminus, the carboxy-terminus orat an internal site. In one embodiment, the peptide compound has atleast one non-β-amyloid peptide-derived amino acid at its N-terminus.Alternatively, the compound can have, for example, 1-3,1-5, 1-7,1-10,1-15 or 1-20 non-β-amyloid peptide-derived amino acid at its N-terminus.In another embodiment, the peptide compound has at least onenon-β-amyloid peptide-derived amino acid at its C-terminus.Alternatively, the compound can have, for example, 1-3,1-5, 1-7,1-10,1-15 or 1-20 non-β-amyloid peptide-derived amino acid at its C-terminus.

[0224] In specific preferred embodiments, an unmodified peptide compoundof the invention comprises Aβ₆₋₂₀ (the amino acid sequence of which isshown in SEQ ID NO: 4), Aβ₁₆₋₃₀ (the amino acid sequence of which isshown in SEQ ID NO: 14), Aβ₁₋₂₀, 26-40 (the amino acid sequence of whichis shown in SEQ ID NO: 15) or EEVVHHHHQQ-βAP₁₆₋₄₀ (the amino acidsequence of which is shown in SEQ ID NO: 16). In the nomenclature usedherein, βAP_(1-20, 26-40) represents βAP₁₋₄₀ in which the internal aminoacid residues 21-25 have been deleted.

[0225] An unmodified peptide compound of the invention can be chemicallysynthesized using standard techniques such as those described inBodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin(1993) and Grant, G. A (ed.). Synthetic Peptides: A User's Guide, W. H.Freeman and Company, New York (1992). Automated peptide synthesizers arecommercially available (e.g., Advanced ChemTech Model 396;Milligen/Biosearch 9600). Alternatively, unmodified peptide compoundscan be prepared according to standard recombinant DNA techniques using anucleic acid molecule encoding the peptide. A nucleotide sequenceencoding the peptide can be determined using the genetic code and anoligonucleotide molecule having this nucleotide sequence can besynthesized by standard DNA synthesis methods (e.g., using an automatedDNA synthesizer). Alternatively, a DNA molecule encoding an unmodifiedpeptide compound can be derived from the natural β-amyloid precursorprotein gene or cDNA (e.g., using the polymerase chain reaction and/orrestriction enzyme digestion) according to standard molecular biologytechniques.

[0226] Accordingly, the invention further provides an isolated nucleicacid molecule comprising a nucleotide sequence encoding a β-amyloidpeptide compound, the β-amyloid peptide compound comprising an aminoacid sequence having at least one amino acid deletion compared toβAP₁₋₃₉ such that the β-amyloid peptide compound alters aggregation ofnatural β-amyloid peptides when contacted with the natural β-amyloidpeptides. As used herein, the term “nucleic acid molecule” is intendedto include DNA molecules and RNA molecules and may be single-stranded ordouble-stranded, but preferably is double-stranded DNA. The isolatednucleic acid encodes a peptide wherein one or more amino acids aredeleted from the N-terminus, C-terminus and/or an internal site ofβAP₁₋₃₉, as discussed above. In yet other embodiments, the isolatednucleic acid encodes a peptide compound having one or more amino acidsdeleted compared to βAP₁₋₃₉ and further having at least one non-p-APderived amino acid residue added to it, for example, at the aminoterminus, the carboxy-terminus or at an internal site. In specificpreferred embodiments, an isolated nucleic acid molecule of theinvention encodes βAP₆₋₂₀, βAP₁₆₋₃₀, βAP_(1-20, 26-40) orEEVVHHHHQQ-βAP₁₆₋₄₀.

[0227] To facilitate expression of a peptide compound in a host cell bystandard recombinant DNA techniques, the isolated nucleic acid encodingthe peptide is incorporated into a recombinant expression vector.Accordingly, the invention also provides recombinant expression vectorscomprising the nucleic acid molecules of the invention. As used herein,the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments may be ligated. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” or simply “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors, which serve equivalent functions.

[0228] In the recombinant expression vectors of the invention, thenucleotide sequence encoding the peptide compound are operatively linkedto one or more regulatory sequences, selected on the basis of the hostcells to be used for expression. The term “operably linked” is intendedto mean that the sequences encoding the peptide compound are linked tothe regulatory sequence(s) in a manner that allows for expression of thepeptide compound. The term “regulatory sequence” is intended to includespromoters, enhancers and other expression control elements (e.g.,polyadenylation signals). Such regulatory sequences are described, forexample, in Goeddel; Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cell, those that direct expression of thenucleotide sequence only in certain host cells (e.g., tissue-specificregulatory sequences) and those that direct expression in a regulatablemanner (e.g., only in the presence of an inducing agent). It will beappreciated by those skilled in the art that the design of theexpression vector may depend on such factors as the choice of the hostcell to be transformed, the level of expression of peptide compounddesired, etc. The expression vectors of the invention can be introducedinto host cells thereby to produce peptide compounds encoded by nucleicacids as described herein.

[0229] The recombinant expression vectors of the invention can bedesigned for expression of peptide compounds in prokaryotic oreukaryotic cells. For example, peptide compounds can be expressed inbacterial cells such as E. coli, insect cells (using baculovirusexpression vectors) yeast cells or mammalian cells. Suitable host cellsare discussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector may be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase. Examples of vectors for expression in yeast S. cerivisaeinclude pYepSec1 (Baldari et al., (1987) EMBO J. 6:229-234), pMFa(Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al.,(1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego,Calif.). Baculovirus vectors available for expression of proteins orpeptides in cultured insect cells (e.g., Sf 9 cells) include the pAcseries (Smith et al., (1983) Mol. Cell. Biol. 3:2156-2165) and the pVLseries (Lucklow, V. A., and Summers, M. D., (1989) Virology 170:31-39).Examples of mammalian expression vectors include pCDM8 (Seed, B., (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40.

[0230] In addition to the regulatory control sequences discussed above,the recombinant expression vector may contain additional nucleotidesequences. For example, the recombinant expression vector may encode aselectable marker gene to identify host cells that have incorporated thevector. Such selectable marker genes are well known in the art.Moreover, the facilitate secretion of the peptide compound from a hostcell, in particular mammalian host cells, the recombinant expressionvector preferably encodes a signal sequence operatively linked tosequences encoding the amino-terminus of the peptide compound such thatupon expression, the peptide compound is synthesized with the signalsequence fused to its amino terminus. This. signal sequence directs thepeptide compound into the secretory pathway of the cell and is thencleaved, allowing for release of the mature peptide compound (i.e., thepeptide compound without the signal sequence) from the host cell. Use ofa signal sequence to facilitate secretion of proteins or peptides frommammalian host cells is well known in the art.

[0231] A recombinant expression vector comprising a nucleic acidencoding a peptide compound that alters aggregation of natural β-AP canbe introduced into a host cell to thereby produce the peptide compoundin the host cell. Accordingly, the invention also provides host cellscontaining the recombinant expression vectors of the invention. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein. A host cell may beany prokaryotic or eukaryotic cell. For example, a peptide compound maybe expressed in bacterial cells such as E. coli, insect cells, yeast ormammalian cells. Preferably, the peptide compound is expressed inmammalian cells. In a preferred embodiment, the peptide compound isexpressed in mammalian cells in vivo in a mammalian subject to treatamyloidosis in the subject through gene therapy (discussed furtherbelow). Preferably, the β-amyloid peptide compound encoded by therecombinant expression vector is secreted from the host cell upon beingexpressed in the host cell.

[0232] Vector DNA can be introduced into prokaryotic or eukaryotic cellsvia conventional transformation or transfection techniques. As usedherein, the terms “transformation” and “transfection” are intended torefer to a variety of art-recognized techniques for introducing foreignnucleic acid (e.g., DNA) into a host cell, including calcium phosphateor calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, electroporation, microinjection andviral-mediated transfection. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratorypress (1989)), and other laboratory manuals. Methods for introducing DNAinto mammalian cells in vivo are also known in the art and can be usedto deliver the vector DNA to a subject for gene therapy purposes(discussed further below).

[0233] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Preferred selectable markers include those that conferresistance to drugs, such as G418, hygromycin and methotrexate. Nucleicacid encoding a selectable marker may be introduced into a host cell onthe same vector as that encoding the peptide compound or may beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

[0234] A nucleic acid of the invention can be delivered to cells in vivousing methods known in the art, such as direct injection of DNA,receptor-mediated DNA uptake or viral-mediated transfection. Directinjection has been used to introduce naked DNA into cells in vivo (seee.g., Acsadi et al. (1991) Nature 332: 815-818; Wolff et al. (1990)Science 247:1465-1468). A delivery apparatus (e.g., a “gene gun”) forinjecting DNA into cells in vivo can be used. Such an apparatus iscommercially available (e.g., from BioRad). Naked DNA can also beintroduced into cells by complexing the DNA to a cation, such aspolylysine, which is coupled to a ligand for a cell-surface receptor(see for example Wu, G. and Wu, C. H. (1988) J. Biol. Chem. 263:14621;Wilson et al. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No.5,166,320). Binding of the DNA-ligand complex to the receptorfacilitates uptake of the DNA by receptor-mediated endocytosis.Additionally, a DNA-ligand complex linked to adenovirus capsids whichnaturally disrupt endosomes, thereby releasing material into thecytoplasm can be used to avoid degradation of the complex byintracellular lysosomes (see for example Curiel et al. (1991) Proc.Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl. Acad.Sci. USA 90:2122-2126).

[0235] Defective retroviruses are well characterized for use in genetransfer for gene therapy purposes (for a review see Miller, A. D.(1990) Blood 76:271). Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound in Current Protocols in Molecular Biology, Ausubel, F. M. et al.(eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 andother standard laboratory manuals. Examples of suitable retrovirusesinclude pLJ, pZIP, pWE and pEM which are well known to those skilled inthe art. Examples of suitable packaging virus lines include ψCrip, ψCre,ψ2 and ψAm. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, endothelialcells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitroand/or in vivo (see for example Eglitis, et al. (1985) Science230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA87:6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573).

[0236] Alternatively, the genome of an adenovirus can be manipulatedsuch that it encodes and expresses a peptide compound but is inactivatedin terms of its ability to replicate in a normal lytic viral life cycle.See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld etal. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell68:143-155. Suitable adenoviral vectors derived from the adenovirusstrain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3,Ad7 etc.) are well known to those skilled in the art. Recombinantadenoviruses are advantageous in that they do not require dividing cellsto be effective gene delivery vehicles and can be used to infect a widevariety of cell types, including airway epithelium (Rosenfeld et al.(1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc.Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993)Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin etal. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584). Additionally,introduced adenoviral DNA (and foreign DNA contained therein) is notintegrated into the genome of a host cell but remains episomal, therebyavoiding potential problems that can occur as a result of insertionalmutagenesis in situations where introduced DNA becomes integrated intothe host genome (e.g., retroviral DNA).

[0237] Adeno-associated virus (AAV) can also be used for delivery of DNAfor gene therapy purposes. AAV is a naturally occurring defective virusthat requires another virus, such as an adenovirus or a herpes virus, asa helper virus for efficient replication and a productive life cycle.(For a review see Muzyczka et al. Curr. Topics in Micro. and Immunol.(1992) 158:97-129). It is also one of the few viruses that may integrateits DNA into non-dividing cells, and exhibits a high frequency of stableintegration (see for example Flotte et al. (1992) Am. J. Respir. Cell.Mol. Biol. 7:349-356; Samulski et al. (1989) J. Virol. 63:3822-3828; andMcLaughlin et al. (1989) J. Virol. 62:1963-1973). Vectors containing aslittle as 300 base pairs of AAV can be packaged and can integrate. AnAAV vector such as that described in Tratschin et al (1985) Mol. Cell.Biol. 5:3251-3260 can be used to introduce DNA into cells. A variety ofnucleic acids have been introduced into different cell types using AAVvectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci.USA 81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081;Wondisford et al. (1988) Mol. Endocrinol. 2:32-39; Tratschin et al.(1984) J. Virol. 51:611-619; and Flotte et al. (1993) J. Biol. Chem.268:3781-3790).

[0238] The invention provides a method for treating a subject for adisorder associated with β-amyloidosis, comprising administering to thesubject a recombinant expression vector encoding a β-amyloid peptidecompound, the compound comprising an amino acid sequence having at leastone amino acid deletion compared to βAP₁₋₃₉, such that the β-amyloidpeptide compound is synthesized in the subject and the subject istreated for a disorder associated with β-amyloidosis. Preferably, thedisorder is Alzheimer's disease. In one embodiment the recombinantexpression vector directs expression of the peptide compound in neuronalcells. In another embodiment, the recombinant expression vector directsexpression of the peptide compound in glial cells. In yet anotherembodiment, the recombinant expression vector directs expression of thepeptide compound in fibroblast cells.

[0239] General methods for gene therapy are known in the art. See forexample, U.S. Pat. No. 5,399,346 by Anderson et al. A biocompatiblecapsule for delivering genetic material is described in PCT PublicationWO 95/05452 by Baetge et al. Methods for grafting genetically modifiedcells to treat central nervous system disorders are described in U.S.Pat. No. 5,082,670 and in PCT Publications WO 90/06757 and WO 93/10234,all by Gage et al. Isolation and/or genetic modification of multipotentneural stem cells or neuro-derived fetal cells are described in PCTPublications WO 94/02593 by Anderson et al., WO 94/16718 by Weiss etal., and WO 94/23754 by Major et al. Fibroblasts transduced with geneticmaterial are described in PCT Publication WO 89/02468 by Mulligan et al.Adenovirus vectors for transfering genetic material into cells of thecentral nervous system are described in PCT Publication WO 94/08026 byKahn et al. Herpes simplex virus vectors suitable for treating neuraldisorders are described in PCT Publications WO 94/04695 by Kaplitt andWO 90/09441 by Geller et al. Promoter elements of the glial fibrillaryacidic protein that can confer astrocyte specific expression on a linkedgene or gene fragment, and which thus can be used for expression of APpeptides specifically in astrocytes, is described in PCT Publication WO93/07280 by Brenner et al. Furthermore, alternative to expression of anAβ peptide to modulate amyloidosis, an antisense oligonucleotide that iscomplementary to a region of the β-amyloid precursor protein mRNAcorresponding to the peptides described herein can be expressed in asubject to modulate amyloidosis. General methods for expressingantisense oligonucleotides to modulate nervous system disorders aredescribed in PCT Publication WO 95/09236.

[0240] Alternative to delivery by gene therapy, a peptide compound ofthe invention comprising an amino acid sequence having at least oneamino acid deletion compared to βAP₁₋₃₉ can be delivered to a subject bydirectly administering the peptide compound to the subject as describedfurther herein for the modified peptide compounds of the invention. Thepeptide compound can be formulated into a pharmaceutical compositioncomprising a therapeutically effective amount of the β-amyloid peptidecompound and a pharmaceutically acceptable carrier. The peptide compoundcan be contacted with natural β-amyloid peptides with a β-amyloidpeptide compound such that aggregation of the natural β-amyloid peptidesis inhibited. Moreover, the peptide compound can be administered to thesubject in a therapeutically effective amount such that the subject istreated for a disorder associated with β-amyloidosis, such asAlzheimer's disease.

[0241] VIII. Other Embodiments

[0242] Although the invention has been illustrated hereinbefore withregard to AP peptide compounds, the principles described, involvingattachment of a modifying group(s) to a peptide compound, are applicableto any amyloidogenic protein or peptide as a means to create a modulatorcompound that modulates, and preferably inhibits, amyloid aggregation.Accordingly, the invention provides modulator compounds that can be usedto treat amyloidosis in a variety of forms and clinical settings.

[0243] Amyloidosis is a general term used to describe pathologicalconditions characterized by the presence of amyloid. Amyloid is ageneral term referring to a group of diverse but specific extracellularprotein deposits which are seen in a number of different diseases.Though diverse in their occurrence, all amyloid deposits have commonmorphologic properties, stain with specific dyes (e.g., Congo red), andhave a characteristic red-green birefringent appearance in polarizedlight after staining. They also share common ultrastructural featuresand common x-ray diffraction and infrared spectra. Amyloidosis can beclassified clinically as primary, secondary, familial and/or isolated.Primary amyloid appears de novo without any preceding disorder.Secondary amyloid is that form which appears as a complication of apreviously existing disorder. Familial amyloid is a geneticallyinherited form found in particular geographic populations. Isolatedforms of amyloid are those that tend to involve a single organ system.

[0244] Different amyloids are characterized by the type of protein(s) orpeptide(s) present in the deposit. For example, as describedhereinbefore, amyloid deposits associated with Alzheimer's diseasecomprise the β-amyloid peptide and thus a modulator compound of theinvention for detecting and/or treating Alzheimer's disease is designedbased on modification of the β-amyloid peptide. The identities of theprotein(s) or peptide(s) present in amyloid deposits associated with anumber of other amyloidogenic diseases have been elucidated.Accordingly, modulator compounds for use in the detection and/ortreatment of these other amyloidogenic diseases can be prepared in asimilar fashion to that described herein for β-AP-derived modulators. Invitro assay systems can be established using an amyloidogenic protein orpeptide which forms fibrils in vitro, analogous to the AP assaysdescribed herein. Modulators can be identified using such assay systems,based on the ability of the modulator to disrupt the β-sheet structureof the fibrils. Initially, an entire amyloidogenic protein can bemodified or, more preferably, a peptide fragment thereof that is knownto form fibrils in vitro can be modified (e.g., analogous to Aβ₁₋₄₀described herein). Amino acid deletion and substitution analyses canthen be performed on the modified protein or peptide (analogous to thestudies described in the Examples) to delineate an aggregation coredomain that is sufficient, when modified, to disrupt fibril formation.

[0245] Non-limiting examples of amyloidogenic proteins or peptides, andtheir associated amyloidogenic disorders, include:

[0246] Transthyretin (TTR)—Amyloids containing transthyretin occur infamilial amyloid polyneuropathy (Portuguese, Japanese and Swedishtypes), familial amyloid cardiomyopathy (Danish type), isolated cardiacamyloid and systemic senile amyloidosis. Peptide fragments oftransthyretin have been shown to form amyloid fibrils in vitro. Forexample, TTR 10-20 and TTR 105-115 form amyloid-like fibrins in 20-30%acetonitrile/water at room temperature (Jarvis, J. A., et al.(1994) Int.J. Pept. Protein Res. 44:388-398). Moreover, familial cardiomyopathy(Danish type) is associated with mutation of Leu at position 111 to Met,and an analogue of TTR 105-115 in which the wildtype Leu at position 111has been substituted with Met (TTR 105-115Met111) also formsamyloid-like fibrils in vitro (see e.g., Hermansen, L. F., et al. (1995)Eur. J. Biochem. 227:772-779; Jarvis et al. supra). Peptide fragments ofTTR that form amyloid fibrils in vitro are also described in Jarvis, J.A., et al. (1993) Biochem. Biophys. Res. Commun. 192:991-998 andGustavsson, A., et al. (1991) Biochem. Biophys. Res. Commun.175:1159-1164. A peptide fragment of wildtype or mutated transthyretinthat forms amyloid fibrils can be modified as described herein to createa modulator of amyloidosis that can be used in the detection ortreatment of familial amyloid polyneuropathy (Portuguese, Japanese andSwedish types), familial amyloid cardiomyopathy (Danish type), isolatedcardiac amyloid or systemic senile amyloidosis.

[0247] Prion Protein (PrP)—Amyloids in a number of spongiformencephalopathies, including scrapie in sheep, bovine spongiformencephalopathy in cows and Creutzfeldt-Jakob disease (CJ) andGerstmann-Straussler-Scheinker syndrome (GSS) in humans, contain PrP.Limited proteolysis of PrPSc (the prion protein associated with scrapie)leads to a 27-30 kDa fragment (PrP27-30) that polymerizes intorod-shaped amyloids (see e.g., Pan, K. M., et al. (1993) Proc. Natl.Acad. Sci. USA 90:10962-10966; Gasset, M., et al. (1993) Proc. Natl.Acad. Sci. USA 90:1-5). Peptide fragments of PrP from humans and othermammals have been shown to form amyloid fibrils in vitro. For example,polypeptides corresponding to sequences encoded by normal and mutantalleles of the PRNP gene (encoding the precursor of the prion proteininvolved in CJ), in the regions of codon 178 and codon 200,spontaneously form amyloid fibrils in vitro (see e.g., Goldfarb, L. G.,et al. (1993) Proc. Natl. Acad. Sci. USA 90:4451-4454). A peptideencompassing residues 106-126 of human PrP has been reported to formstraight fibrils similar to those extracted from GSS brains, whereas apeptide encompassing residues 127-147 of human PrP has been reported toform twisted fibrils resembling scrapie-associated fibrils (Tagliavini,F., et al. (1993) Proc. Natl. Acad. Sci. USA 90:9678-9682). Peptides ofSyrian hamster PrP-encompassing residues 109-122, 113-127, 113-120,178-191 or 202-218 have been reported to form amyloid fibrils, with themost amyloidogenic peptide being Ala-Gly-Ala-Ala-Ala-Ala-Gly-Ala (SEQ IDNO: 17), which corresponds to residues 113-120 of Syrian hamster PrP butwhich is also conserved in PrP from other species (Gasset, M., et al.(1992) Proc. Natl. Acad. Sci. USA 89:10940-10944). A peptide fragment ofPrP that forms amyloid fibrils can be modified as described herein tocreate a modulator of amyloidosis that can be used in the detection ortreatment of scrapie, bovine spongiform encephalopathy,Creutzfeldt-Jakob disease or Gerstmann-Straussler-Scheinker syndrome.

[0248] Islet Amyloid Polypeptide (IAPP, also known as amylin)—Amyloidscontaining IAPP occur in adult onset diabetes and insulinoma. IAPP is a37 amino acid polypeptide formed from an 89 amino acid precursor protein(see e.g., Betsholtz, C., et al. (1989) Exp. Cell. Res. 183:484-493;Westermark, P., et al. (1987) Proc. Natl. Acad. Sci. USA 84:3881-3885).A peptide corresponding to IAPP residues 20-29 has been reported to formamyloid-like fibrils in vitro, with residues 25-29, having the sequenceAla-Ile-Leu-Ser-Ser (SEQ ID NO: 18), being strongly amyloidogenic(Westermark, P., et al. (1990) Proc. Natl. Acad. Sci. USA 87:5036-5040;Glenner, G. G., et al. (1988) Biochem. Biophys. Res. Commun.155:608-614). A peptide fragment of IAPP that forms amyloid fibrils canbe modified as described herein to create a modulator of amyloidosisthat can be used in the detection or treatment of adult onset diabetesor insulinoma.

[0249] Atrial Natriuretic Factor (ANF)—Amyloids containing ANF areassociated with isolated atrial amyloid (see e.g., Johansson, B., et al.(1987) Biochem. Biophys. Res. Commun. 148:1087-1092). ANF corresponds toamino acid residues 99-126 (proANF99-126) of the ANF prohormone(proANPI-126) (Pucci, A., et al. (1991) J. Pathol. 165:235-241). ANF, ora fragment thereof, that forms amyloid fibrils can be modified asdescribed herein to create a modulator of amyloidosis that can be usedin the detection or treatment of isolated atrial amyloid.

[0250] Kappa or Lambda Light Chain—Amyloids containing kappa or lambdalight chains are associated idiopathic (primary) amyloidosis, myeloma ormacroglobulinemia-associated amyloidosis, and primary localizedcutaneous nodular amyloidosis associated with Sjogren's syndrome. Thestructure of amyloidogenic kappa and lambda light chains, includingamino acid sequence analysis, has been characterized (see e.g., Buxbaum,J. N., et al. (1990) Ann. Intern. Med. 112:455-464; Schormann, N., etal. (1995) Proc. Natl. Acad. Sci. USA 92:9490-9494; Hurle, M. R., et al.(1994) Proc. Natl. Acad. Sci. USA 91:5446-5450; Liepnieks, J. J., et al.(1990) Mol. Immunol. 27:481-485; Gertz, M. A., et al. (1985) Scand. J.Immunol. 22:245-250; Inazumi, T., et al. (1994) Dermatology189:125-128). Kappa or lambda light chains, or a peptide fragmentthereof that forms amyloid fibrils, can be modified as described hereinto create a modulator of amyloidosis that can be used in the detectionor treatment of idiopathic (primary) amyloidosis, myeloma ormacroglobulinemia-associated amyloidosis or primary localized cutaneousnodular amyloidosis associated with Sjogren's syndrome.

[0251] Amyloid A—Amyloids containing the amyloid A protein (AA protein),derived from serum amyloid A, are associated with reactive (secondary)amyloidosis (see e.g., Liepnieks, J. J., et al. (1995) Biochim. Biophys.Acta 1270:81-86), familial Mediterranean Fever and familial amyloidnephropathy with urticaria and deafness (Muckle-Wells syndrome) (seee.g., Linke, R. P., et al. (1983) Lab. Invest. 48:698-704). Recombinanthuman serum amyloid A forms amyloid-like fibrils in vitro (Yamada, T.,et al. (1994) Biochim. Biophys. Acta 1226:323-329) and circulardichroism studies revealed a predominant β sheet/turn structure(McCubbin, W. D., et al. (1988) Biochem J. 256:775-783). Serum amyloidA, amyloid A protein or a fragment thereof that forms amyloid fibrilscan be modified as described herein to create a modulator of amyloidosisthat can be used in the detection or treatment of reactive (secondary)amyloidosis, familial Mediterranean Fever and familial amyloidnephropathy with urticaria and deafness (Muckle-Wells syndrome).

[0252] Cystatin C—Amyloids containing a variant of cystatin C areassociated with hereditary cerebral hemorrhage with amyloidosis ofIcelandic type. The disease is associated with a leucine to glycinemutation at position 68 and cystatin C containing this mutationaggregates in vitro (Abrahamson, M. and Grubb, A. (1994) Proc. Natl.Acad. Sci. USA 91:1416-1420). Cystatin C or a peptide fragment thereofthat forms amyloid fibrils can be modified as described herein to createa modulator of amyloidosis that can be used in the detection ortreatment of hereditary cerebral hemorrhage with amyloidosis ofIcelandic type.

[0253] β2 microglobulin—Amyloids containing β2 microglobulin (β2M) are amajor complication of long term hemodialysis (see e.g., Stein, G., etal. (1994) Nephrol. Dial. Transplant. 9:48-50; Floege, J., et al. (1992)Kidney Int. Suppl. 38:S78-S85; Maury, C. P. (1990) Rheumatol. Int.10:1-8). The native β2M protein has been shown to form amyloid fibrilsin vitro (Connors, L. H., et al. (1985) Biochem. Biophys. Res. Commun.131:1063-1068; Ono, K., et al. (1994) Nephron 66:404-407). β2M, or apeptide fragment thereof that forms amyloid fibrils, can be modified asdescribed herein to create a modulator of amyloidosis that can be usedin the detection or treatment of amyloidosis associated with long termhemodialysis.

[0254] Apolipoprotein A-I (ApoA-I)—Amyloids containing variant forms ofApoA-I have been found in hereditary non-neuropathic systemicamyloidosis (familial amyloid polyneuropathy III). For example,N-terminal fragments (residues 1-86, 1-92 and 1-93) of an ApoA-I varianthaving a Trp to Arg mutation at position 50 have been detected inamyloids (Booth, D. R., et al. (1995) QJM 88:695-702). In anotherfamily, a leucine to arginine mutation at position 60 was found (Soutar,A. K., et al. (1992) Proc. Natl. Acad. Sci. USA 89:7389-7393). ApoA-I ora peptide fragment thereof that forms amyloid fibrils can be modified asdescribed herein to create a modulator of amyloidosis that can be usedin the detection or treatment of hereditary non-neuropathic systemicamyloidosis.

[0255] Gelsolin—Amyloids containing variants of gelsolin are associatedwith familial amyloidosis of Finnish type. Synthetic gelsolin peptidesthat have sequence homology to wildtype or mutant gelsolins and thatform amyloid fibrils in vitro are reported in Maury, C. P. et al. (1994)Lab. Invest. 70:558-564. A nine residue segment surrounding residue 187(which is mutated in familial gelsolin amyloidosis) was defined as anamyloidogenic region (Maury, et al., supra; see also Maury, C. P., etal. (1992) Biochem. Biophys. Res. Commun. 183:227-231; Maury, C. P.(1991) J. Clin. Invest. 87:1195-1199). Gelsolin or a peptide fragmentthereof that forms amyloid fibrils can be modified as described hereinto create a modulator of amyloidosis that can be used in the detectionor treatment of familial amyloidosis of Finnish type.

[0256] Procalcitonin or calcitonin—Amyloids containing procalcitonin,calcitonin or calcitonin-like immunoreactivity have been detected inamyloid fibrils associated with medullary carcinoma of the thyroid (seee.g., Butler, M. and Khan, S. (1986) Arch. Pathol. Lab. Med.110:647-649; Sletten, K., et al. (1976) J. Exp. Med. 143:993-998).Calcitonin has been shown to form a nonbranching fibrillar structure invitro (Kedar, I., et al. (1976) Isr. J. Med. Sci. 12:1137-1140).Procalcitonin, calcitonin or a fragment thereof that forms amyloidfibrils can be modified as described herein to create a modulator ofamyloidosis that can be used in the detection or treatment ofamyloidosis associated with medullary carcinoma of the thyroid.

[0257] Fibrinogen—Amyloids containing a variant form of fibrinogenalpha-chain have been found in hereditary renal amyloidosis. An arginineto leucine mutation at position 554 has been reported in amyloid fibrilprotein isolated from postmortem kidney of an affected individual(Benson, M. D., et al. (1993) Nature Genetics 3:252-255). Fibrinogenalpha-chain or a peptide fragment thereof that forms amyloid fibrils canbe modified as described herein to create a modulator of amyloidosisthat can be used in the detection or treatment of fibrinogen-associatedhereditary renal amyloidosis.

[0258] Lysozyme—Amyloids containing a variant form of lysozyme have beenfound in hereditary systemic amyloidosis. In one family the disease wasassociated with a threonine to isoleucine mutation at position 56,whereas in another family the disease was associated with a histidine toaspartic acid mutation at position 67 (Pepys, M. B., et al. (1993)Nature 362:553-557). Lysozyme or a peptide fragment thereof that formsamyloid fibrils can be modified as described herein to create amodulator of amyloidosis that can be used in the detection or treatmentof lysozyme-associated hereditary systemic amyloidosis.

[0259] This invention is further illustrated by the following exampleswhich should not be construed as limiting. A modulator's ability toalter the aggregation of β-amyloid peptide in the assays described beloware predictive of the modulator's ability to perform the same functionin vivo. The contents of all references, patents and published patentapplications cited throughout this application are hereby incorporatedby reference.

EXAMPLE 1 Construction of β-Amyloid Modulators

[0260] A β-amyloid modulator composed of an amino-terminallybiotinylated β-amyloid peptide of the amino acid sequence:

[0261] DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV

[0262] (positions 1 to 40 of SEQ ID NO: 1) was prepared by solid-phasepeptide synthesis using an N^(α)-9-fluorenylmethyloxycarbonyl(FMOC)-based protection strategy as follows. Starting with 2.5 mmoles ofFMOC-Val-Wang resin, sequential additions of each amino acid wereperformed using a four-fold excess of protected amino acids,1-hydroxybenzotriazole (HOBt) and diisopropyl carbodiimide (DIC).Recouplings were performed when necessary as determined by ninhydrintesting of the resin after coupling. Each synthesis cycle was minimallydescribed by a three minute deprotection (25%piperidine/N-methyl-pyrrolidone (NMP)), a 15 minute deprotection, fiveone minute NMP washes, a 60 minute coupling cycle, five NMP washes and aninhydrin test. To a 700 mg portion of the fully assembledpeptide-resin, biotin (obtained commercially from Molecular Probes,Inc.) was substituted for an FMOC-amino acid was coupled by the aboveprotocol. The peptide was removed from the resin by treatment withtrifluoroacetic acid (TFA) (82.5%), water (5%), thioanisole (5%), phenol(5%), ethanedithiol (2.5%) for two hours followed by precipitation ofthe peptide in cold ether. The solid was pelleted by centrifugation(2400 rpm×10 min.), and the ether decanted. It was resuspended in ether,pelleted and decanted a second time. The solid was dissolved in 10%acetic acid and lyophilized to dryness to yield 230 mg of crudebiotinylated peptide. 60 mg of the solid was dissolved in 25%acetonitrile (ACN)/0.1% TFA and applied to a C18 reversed phase highperformance liquid chromatography (HPLC) column. Biotinyl βAP₁₋₄₀ waseluted using a linear gradient of 30-45% acetonitrile/0.1% TFA over 40minutes. One primary fraction (4 mg) and several side fractions wereisolated. The main fraction yielded a mass spectrum of 4556(matrix-assisted laser desorption ionization—time of flight) whichmatches the theoretical (4555) for this peptide.

[0263] A β-amyloid modulator composed of an amino-terminallybiotinylated β-amyloid peptide of the amino acid sequence:

[0264] DAEFRHDSGYEVHHQ

[0265] (positions 1 to 15 of SEQ ID NO: 1) was prepared on an AdvancedChemTech Model 396 multiple peptide synthesizer using an automatedprotocol established by the manufacturer for 0.025 mmole scalesynthesis. Double couplings were performed on all cycles using2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU)/N,N-diisopropylethylamine (DIEA)/HOBt/FMOC-AA in four-fold excessfor 30 minutes followed by DIC/HOBt/FMOC-AA in four-fold excess for 45minutes. The peptide was deprotected and removed from the resin bytreatment with TFA/water (95%/5%) for three hours and precipitated withether as described above. The pellet was resuspended in 10% acetic acidand lyophilized. The material was purified by a preparative HPLC using15%-40% acetonitrile over 80 minutes on a Vydac C18 column (21×250 mm).The main isolate eluted as a single symmetrical peak when analyzed byanalytical HPLC and yielded the expected molecular weight when analyzedby electrospray mass spectrometry. Result=2052.6 (2052 theoretical).

[0266] β-amyloid modulator compounds comprising other regions of theβ-AP amino acid sequence (e.g., an Aβ aggregation core domain) weresimilarly prepared using the synthesis methods described above.Moreover, modulators comprising other amyloidogenic peptides can besimilarly prepared.

EXAMPLE 2 Inhibition of β-Amyloid Aggregation by Modulators

[0267] The ability of β-amyloid modulators to inhibit the aggregation ofnatural β-AP when combined with the natural β-AP was examined in aseries of aggregation assays. Natural β-AP(β-AP₁₋₄₀) was obtainedcommercially from Bachem (Torrance, Calif.). Amino-terminallybiotinylated β-AP modulators were prepared as described in Example 1.

[0268] A. Optical Density Assay

[0269] In one assay, β-AP aggregation was measured by determining theincrease in turbidity of a solution of natural β-AP over time in theabsence or presence of various concentrations of the modulator.Turbidity of the solution was quantitated by determining the opticaldensity at 400 nm (A_(400 nm)) of the solution over time.

[0270] The aggregation of natural β-AP in the absence of modulator wasdetermined as follows. βAP₁₋₄₀ was dissolved in hexafluoro isopropanol(HFIP; Aldrich Chemical Co., Inc.) at 2 mg/ml. Aliquots of the HFIPsolution (87 ill) were transferred to individual 10 mm×75 mm test tubes.A stream of argon gas was passed through each tube to evaporate theHFIP. To the resulting thin film of peptide, dimethylsulfoxide (DMSO;Aldrich Chemical Co., Inc.) (25 μl) was added to dissolve the peptide. A2 mm×7 mm teflon-coated magnetic stir bar was added to each tube. Buffer(475 μL of 100 mM NaCl, 10 mM sodium phosphate, pH 7.4) was added to theDMSO solution with stirring. The resulting mixture was stirredcontinuously and the optical density was monitored at 400 nm to observethe formation of insoluble peptide aggregates.

[0271] Alternatively, β-AP₁₋₄₀ was dissolved in DMSO as described aboveat 1.6 mM (6.9 mg/ml) and aliquots (25 μl) were added to stirred buffer(475 el), followed by monitoring of absorbance at 400 nm.

[0272] For inhibition studies in which a β-amyloid modulator wasdissolved in solution together with the natural β-AP, the modulatorswere dissolved in DMSO either with or without prior dissolution in HFIP.These compounds were then added to buffer with stirring, followed byaddition of β-AP₁₋₄₀ in DMSO. Alternatively, HFIP solutions ofmodulators were combined with β-AP₁₋₄₀ in HFIP followed by evaporationand redissolution of the mixture in DMSO. Buffer was then added to theDMSO solution to initiate the assay. The amino-terminally biotinylatedβ-amyloid peptide modulators N-biotinyl-βAP₁₋₄₀, and N-biotinyl-βAP₁₋₁₅were tested at concentrations of 1% and 5% in the natural β-AP₁₋₄₀solution.

[0273] A representative example of the results is shown graphically inFIG. 1, which depicts the inhibition of aggregation of natural β-AP₁₋₄₀by N-biotinyl-βAP₁₋₄₀. In the absence of the modulator, the opticaldensity of the natural β-AP solution showed a characteristic sigmoidalcurve, with a lag time prior to aggregation (approximately 3 hours inFIG. 1) in which the A_(400 nm) was low, followed by rapid increase inthe A_(400 nm), which quickly reached a plateau level, representingaggregation of the natural β amyloid peptides. In contrast, in thepresence of as little as 1% of the N-biotinyl-βAP₁₄₀ modulator,aggregation of the natural β amyloid peptides was markedly inhibited,indicated by an increase in the lag time, a decrease in the slope ofaggregation and a decrease in the plateau level reached for theturbidity of the solution (see FIG. 1). N-biotinyl-βAP₁₋₄₀ at aconcentration of 5% similarly inhibited aggregation of the natural βamyloid peptide. Furthermore, similar results were observed whenN-biotinyl-βAP₁₋₁₅ was used as the modulator. These results demonstratethat an N-terminally biotinylated β-AP modulator can effectively inhibitthe aggregation of natural β amyloid peptides, even when the natural pamyloid peptides are present at as much as a 100-fold molar excessconcentration.

[0274] B. Fluorescence Assay

[0275] In a second assay, β-AP aggregation was measured using afluorometric assay essentially as described in Levine, H. (1993) ProteinScience 2:404-410. In this assay, the dye thioflavine T (ThT) iscontacted with the β-AP solution. Association of ThT with aggregatedβ-AP, but not monomeric or loosely associated β-AP, gives rise to a newexcitation (ex) maximum at 450 nm and an enhanced emission (em) at 482nm, compared to the 385 nm (ex) and 445 nm (em) for the free dye. β-APaggregation was assayed by this method as follows. Aliquots (2.9 μl) ofthe solutions used in the aggregation assays as described above insection A were removed from the samples and diluted in 200 μl ofpotassium phosphate buffer (50 mM, pH 7.0) containing thioflavin T (10μM; obtained commercially from Aldrich Chemical Co., Inc.). Excitationwas set at 450 nm and emission was measured at 482 mm. Similar to theresults observed with the optical density assay described above insection A, as little as 1% of the N-biotinylated β-AP modulators waseffective at inhibiting the aggregation of natural β amyloid peptidesusing this fluorometric assay.

[0276] C. Static Aggregation Assay

[0277] In a third assay, β-AP aggregation was measured by visualizationof the peptide aggregates using SDS-polyacrylamide gel electrophoresis(SDS-PAGE). In this assay, β-AP solutions were allowed to aggregate overa period of time and then aliquots of the reaction were run on astandard SDS-PAGE gel. Typical solution conditions were 200 μM ofβ-AP₁₋₄₀ in PBS at 37° C. for 8 days or 200 μM β-AP₁₋₄₀ in 0.1 M sodiumacetate at 37° C. for 3 days. The peptide aggregates were visualized byCoomassie blue staining of the gel or, for β-AP solutions that includeda biotinylated S-AP modulator, by western blotting of a filter preparedfrom the gel with a streptavidin-peroxidase probe, followed by astandard peroxidase assay. The β-AP aggregates are identifiable as highmolecular weight, low mobility bands on the gel, which are readilydistinguishable from the low molecular weight, high mobility β-APmonomer or dimer bands.

[0278] When natural β-AP₁₋₄₀ aggregation was assayed by this method inthe absence of any β amyloid modulators, high molecular weightaggregates were readily detectable on the gel. In contrast, whenN-biotinyl-β-AP₁₋₄₀ modulator self-aggregation was assayed (i.e.,aggregation of the N-biotinyl peptide alone, in the absence of anynatural β-AP), few if any high molecular weight aggregates wereobserved, indicating that the ability of the modulator to self-aggregateis significantly reduced compared to natural β-AP. Finally, whenaggregation of a mixture of natural β-AP₁₋₄₀ and N-biotinylated β-AP₁₋₄₀was assayed by this method, reduced amounts of the peptide mixtureassociated into high molecular weight aggregates, thus demonstratingthat the β amyloid modulator is effective at inhibiting the aggregationof the natural β amyloid peptides.

EXAMPLE 3 Neurotoxicity Analysis of β-Amyloid Modulators

[0279] The neurotoxicity of the β-amyloid modulators is tested in acell-based assay using the neuronal precursor cell line PC-12, orprimary neuronal cells, and the viability indicator3,(4,4-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT). (SeeShearman, M. S. et al. (1994) Proc. Natl. Acad. Sci. USA 91:1470-1474;Hansen, M. B. et al. (1989) J. Immun. Methods 119:203-210). PC-12 is arat adrenal pheochromocytoma cell line and is available from theAmerican Type Culture Collection, Rockville, Md. (ATCC CRL 1721). MTT(commercially available from Sigma Chemical Co.) is a chromogenicsubstrate that is converted from yellow to blue in viable cells, whichcan be detected spectrophotometrically.

[0280] To test the neurotoxicity of a β-amyloid modulator (either aloneor combined with natural β-AP), cells first are plated in 96-well platesat 7,000-10,000 cells/well and allowed to adhere by overnight culture at37° C. Serial dilutions of freshly dissolved or “aged” modulators(either alone or combined with natural N-AP) in phosphate bufferedsaline (PBS) are added to the wells in triplicate and incubation iscontinued for two or more days. Aged modulators are prepared byincubating an aqueous solution of the modulator at 37° C. undisturbedfor a prolonged period (e.g., five days or more). For the final twohours of exposure of the cells to the modulator preparation, MTT isadded to the media to a final concentration of 1 mg/ml and incubation iscontinued at 37° C. Following the two hour incubation with MTT, themedia is removed and the cells are lysed in isopropanol/0.4N HCl withagitation. An equal volume of PBS is added to each well and theabsorbance of each well at 570 nm is measured to quantitate viablecells. Alternatively, MTT is solubilized by addition of 50% N,N-dimethylformamide/20% sodium dodecyl sulfate added directly to the media in thewells and viable cells are likewise quantitated by measuring absorbanceat 570 nm. The relative neurotoxicity of a β-amyloid modulator (eitheralone or in combination with natural β-AP) is determined by comparisonto natural β-AP alone (e.g., β1-40, β1-42), which exhibits neurotoxicityin this assay and thus can serve as a positive control.

EXAMPLE 4 Syntheses of Additional Modified β-Amyloid Peptide Compounds

[0281] In this example, a series of modified β-APs, having a variety ofN-terminal or random side chain modifications were synthesized.

[0282] A series of N-terminally modified p-amyloid peptides wassynthesized using standard methods. Fully-protected resin-bound peptidescorresponding to Aβ(1-15) and Aβ(1-40) were prepared as described inExample 1 on Wang resin to eventually afford carboxyl terminal peptideacids. Small portions of each peptide resin (13 and 20 μmoles,respectively) were aliquoted into the wells of the reaction block of anAdvanced ChemTech Model 396 Multiple Peptide Synthesizer. The N-terminalFMOC protecting group of each sample was removed in the standard mannerwith 25% piperidine in NMP followed by extensive washing with NMP. Theunprotected N-terminal a-amino group of each peptide-resin sample wasmodified using one of the following methods:

[0283] Method A, coupling of modifying reagents containing freecarboxylic acid groups: The modifying reagent (five equivalents) waspredissolved in NMP, DMSO or a mixture of these two solvents. HOBT andDIC (five equivalents of each reagent) were added to the dissolvedmodifier and the resulting solution was added to one equivalent offree-amino peptide-resin. Coupling was allowed to proceed overnight,followed by washing. If a ninhydrin test on a small sample ofpeptide-resin showed that coupling was not complete, the coupling wasrepeated using 1-hydroxy-7-azabenzotriazole (HOAt) in place of HOBt.

[0284] Method B, coupling of modifying reagents obtained in preactivatedforms: The modifying reagent (five equivalents) was predissolved in NMP,DMSO or a mixture of these two solvents and added to one equivalent ofpeptide-resin. Diisopropylethylamine (DIEA; six equivalents) was addedto the suspension of activated modifier and peptide-resin. Coupling wasallowed to proceed overnight, followed by washing. If a ninhydrin teston a small sample of peptide-resin showed that coupling was notcomplete, the coupling was repeated.

[0285] After the second coupling (if required) the N-terminally modifiedpeptide-resins were dried at reduced pressure and cleaved from the resinwith removal of side-chain protecting groups as described in Example 1.Analytical reversed-phase HPLC was used to confirm that a major productwas present in the resulting crude peptides which were purified usingMillipore Sep-Pak cartridges or preparative reverse-phase HPLC. Massspectrometry was used to confirm the presence of the desired compound inthe product.

[0286] Method A was used to couple N-acetylneuraminic acid, cholic acid,trans-4-cotininecarboxylic acid, 2-imino-1-imidazolidineacetic acid,(S)-(−)-indoline-2-carboxylic acid, (−)-menthoxyacetic acid,2-norbornaneacetic acid, γ-oxo-5-acenaphthenebutyric acid,(−)-2-oxo-4-thiazolidinecarboxylic acid, and tetrahydro-3-furoic acid.Method B was used to couple 2-iminobiotin-N-hydroxysuccinimide ester,diethylenetriaminepentaacetic dianhydride, 4-morpholinecarbonylchloride, 2-thiopheneacetyl chloride, and 2-thiophenesulfonyl chloride.

[0287] In a manner similar to the construction of N-terminally modifiedAβ(1-15) and Aβ(1-40) peptides described above, N-fluoresceinyl Aβ(1-15)and Aβ(1-40) were prepared in two alternative manners using thepreactivated reagents 5-(and 6)-carboxyfluorescein succinimidyl esterand fluorescein-5-isothiocyanate (FITC Isomer I). Both reagents wereobtained from Molecular Probes Inc. Couplings were performed using fourequivalents of reagent per equivalent of peptide-resin with DIEA addedto make the reaction solution basic to wet pH paper. Couplings of eachreagent to Aβ(1-15)-resin appeared to be complete after a singleovernight coupling. Coupling to Aβ(1-40)-resin was slower as indicatedby a positive ninhydrin test and both reagents were recoupled to thispeptide-resin overnight in tetrahydrofuran-NMP (1:2 v/v). The resultingN-terminally modified peptide-resins were cleaved, deprotected andpurified as described in Example A.

[0288] In addition to the N-fluoresceinyl Aβ peptides described above, aβ-amyloid modulator comprised of random modification of Aβ(1-40) withfluorescein was prepared. Aβ(1-40) purchased from Bachem was dissolvedin DMSO at approximately 2 mg/mL. 5-(and-6)-Carboxyfluorescein purchasedfrom Molecular Probes was added in a 1.5 molar excess and DIEA was addedto make the solution basic to wet pH paper. The reaction was allowed toproceed for 1 hour at room temperature and was then quenched withtriethanolamine. The product was added to assays as this crude mixture.

[0289] β-amyloid modulator compounds comprising other regions of theβ-AP amino acid sequence (e.g., an AP aggregation core domain) weresimilarly prepared using the synthesis methods described above.Moreover, modulators comprising other amyloidogenic peptides can besimilarly prepared.

EXAMPLE 5 Identification of Additional β-Amyloid Modulators

[0290] In this Example, two assays of Aβ aggregation were used toidentify β-amyloid modulators which can inhibit this process.

[0291] The first assay is referred to as a seeded static assay (SSA) andwas performed as follows:

[0292] To prepare a solution of Aβ monomer, the appropriate quantity ofAβ(1-40) peptide (Bachem) was weighed out on a micro-balance (the amountwas corrected for the amount of water in the preparation, which,depending on lot number, was 20-30% w/w). The peptide was dissolved in{fraction (1/25)} volume of dimethysulfoxide (DMSO), followed by waterto ½ volume and ½ volume 2×PBS (10×PBS: NaCl 137 mM, KCl 2.7 mMNa₂HPO₄.7H₂O 4.3 mM, KH₂PO₄ 1.4 mM pH 7.2) to a final concentration of200 μM.

[0293] To prepare a stock seed, 1 ml of the above Aβ monomerpreparation, was incubated for 8 days at 37° C. and sheared sequentiallythrough an 18, 23, 26 and 30 gauge needle 25, 25, 50, and 100 timesrespectively. 2 μl samples of the sheared material was taken forfluorescence measurements after every 50 passes through the 30 gaugeneedle until the fluorescence units (FU) had plateaued (approx.100-150×).

[0294] To prepare a candidate inhibitor, the required amount ofcandidate inhibitor was weighed out and the stock dissolved in 1×PBS toa final concentration of 1 mM (10× stock). If insoluble, it wasdissolved in {fraction (1/10)} volume of DMSO and diluted in 1×PBS to 1mM. A further {fraction (1/10)} dilution was also prepared to test eachcandidate at both 100 μM and 10 μM.

[0295] For the aggregation assay, each sample was set up in triplicate[50 μl of 200 μM monomer, 125 FU sheared seed (variable quantitydependent on the batch of seed, routinely 3-6 μl), 10 μl of 10×inhibitor solution, final volume made up to 100 μl with 1×PBS]. Twoconcentrations of each inhibitor were tested 100 μM and 10 μM,equivalent to a 1:1 and a 1:10 molar ratio of monomer to inhibitor. Thecontrols included an unseeded reaction to confirm that the fresh monomercontained no seed, and a seeded reaction in the absence of inhibitor, asa reference to compare against putative inhibitors. The assay wasincubated at 37° C. for 6 h, taking 2 μl samples hourly for fluorescencemeasurements. To measure fluorescence, a 2 μl sample of Aβ was added to400 μl of Thioflavin-T solution (50 mM Potassium Phosphate 10 mMThioflavin-T pH 7.5). The samples were vortexed and the fluorescence wasread in a 0.5 ml micro quartz cuvette at EX 450 nm and EM 482 nm(Hitachi 4500 Fluorimeter). β-aggregation results in enhanced emissionof Thioflavin-T. Accordingly, samples including an effective inhibitorcompound exhibit reduced emission as compared to control samples withoutthe inhibitor compound.

[0296] The second assay is referred to as a shaken plate aggregationassay and was performed as follows:

[0297] Aβ(1-40) peptide from Bachem (Torrance, Calif.) was dissolved inHFIP (1,1,1,3,3,3-Hexafluoro-2-propanol; Aldrich 10,522-8) at aconcentration of 2 mg peptide/ml and incubated at room temperature for30 min. HFIP solubilized peptide was sonicated in a waterbath sonicatorfor 5 min at highest setting, then evaporated to dryness under a streamof argon. The peptide film was resuspended in anhydrousdimethylsulfoxide (DMSO) at a concentration of 6.9 mg/ml, sonicated for5 min as before, then filtered through a 0.2 micron nylon syringe filter(VWR cat. No. 28196-050). Candidate inhibitors were dissolved directlyin DMSO, generally at a molar concentration 4 times that of the Aβ(1-40)peptide.

[0298] Candidates were assayed in triplicate. For each candidate to betested, 4 parts Aβ(1-40) peptide in DMSO were combined with 1 partcandidate inhibitor in DMSO in a glass vial, and mixed to produce a 1:1molar ratio of Aβ peptide to candidate. For different molar ratios,candidates were diluted with DMSO prior to addition to Aβ(1-40), inorder to keep the final DMSO and Aβ(1-40) concentrations constant. Intoan ultra low binding 96 well plate (Corning Costar cat. No. 2500,Cambridge Mass.) 100 μl PTL buffer (150 mM NaCl, 10 mM NaH₂PO₄; pH 7.4)was aliquotted per well. For each candidate, 10 μl of peptide mixture inDMSO was aliquotted into each of three wells containing buffer. Thecovered plate was vigorously vortexed on a plate shaker at high speedfor 30 seconds. An additional 100 μl of PTL buffer was added to eachwell and again the plate was vortexed vigorously for 30 sec. Absorbanceat 405 nm was immediately read in a plate reader for a baseline reading.The plate was returned to the plate shaker and vortexed at moderatespeed for 5 hours at room temperature, with absorbance readings taken at15-20 min intervals. Increased absorbance indicated aggregation.Accordingly, effective inhibitor compounds cause a decrease inabsorbance in the test sample as compared to a control sample withoutthe inhibitor compound.

[0299] Representative results of the static seeded assay and shakenplate assay with preferred β-amyloid modulators are shown below in TableI. TABLE I Effect Effect in in Seeded Candidate Aβ Amino Modifyingshaken plate Static Inhibitor Acids Reagent assay Assay* 174 Aβ1-15Cholic acid Complete ++ inhibition at 100% conc 176 Aβ1-15 Diethylene-Decreased ++ triamine penta Plateau acetic acid 180 Aβ1-15 (−)-MenthoxyNone ++ acetic acid 190 Aβ1-15 Fluorescein Decreased ++ carboxylic acidPlateau (FICO) 220 Aβ16-40 h-EVHHHHQQK- Complete ++ mutant [Aβ(16-40)]-OH inhibition at 100%, increased lag at 10% 224 Aβ1-40F₁₉F₂₀−>T₁₉T₂₀ Increased lag ++ mutant 233 A6β-20 Acetic acidaccelerated ++ aggregation at 10% conc

[0300] These results indicate that β-APs modified by a wide variety ofN-terminal modifying groups are effective at modulating β-amyloidaggregation.

EXAMPLE 6 Additional β-Amyloid Aggregation Assays

[0301] Most preferably, the ability of β-amyloid modulator compounds tomodulate (e.g., inhibit or promote) the aggregation of natural β-AP whencombined with the natural β-AP is examined in one or both of theaggregation assays described below. Natural β-AP (β-AP₁₋₄₀) for use inthe aggregation assays is commercially available from Bachem (Torrance,Calif.).

[0302] A. Nucleation Assay

[0303] The nucleation assay is employed to determine the ability of testcompounds to alter (e.g. inhibit) the early events in formation of β-APfibers from monomeric β-AP. Characteristic of a nucleated polymerizationmechanism, a lag time is observed prior to nucleation, after which thepeptide rapidly forms fibers as reflected in a linear rise in turbidity.The time delay before polymerization of β-AP monomer can be quantifiedas well as the extent of formation of insoluble fiber by lightscattering (turbidity). Polymerization reaches equilibrium when themaximum turbidity reaches a plateau. The turbidity of a solution ofnatural β-AP in the absence or presence of various concentrations of aβ-amyloid modulator compound is determined by measuring the apparentabsorbance of the solution at 405 nm (A_(405 nm)) over time. Thethreshold of sensitivity for the measurement of turbidity is in therange of 15-20 μM β-AP. A decrease in turbidity over time in thepresence of the modulator, as compared to the turbidity in the absenceof the modulator, indicates that the modulator inhibits formation ofβ-AP fibers from monomeric β-AP. This assay can be performed usingstirring or shaking to accelerate polymerization, thereby increasing thespeed of the assay. Moreover the assay can be adapted to a 96-well plateformat to screen multiple compounds.

[0304] To perform. the nucleation assay, first Aβ₁₋₄₀ peptide isdissolved in HFIP (1,1,1,3,3,3-Hexafluoro-2-propanol; Aldrich 10,522-8)at a concentration of 2 mg peptide/ml and incubated at room temperaturefor 30 min. HFIP-solubilized peptide is sonicated in a waterbathsonicator for 5 min at highest setting, then evaporated to dryness undera stream of argon. The peptide film is resuspended in anhydrousdimethylsulfoxide (DMSO) at a concentration of 6.9 mg/ml(25×concentration), sonicated for 5 min as before, then filtered througha 0.2 micron nylon syringe filter (VWR cat. No. 28196-050). Testcompounds are dissolved in DMSO at a 100× concentration. Four volumes of25×Aβ₁₋₄₀ peptide in DMSO are combined with one volume of test compoundin DMSO in a glass vial, and mixed to produce a 1:1 molar ratio of APpeptide to test compound. For different molar ratios, test compounds arediluted with DMSO prior to addition to Aβ₁₋₄₀, in order to keep thefinal DMSO and Aβ₁₋₄₀ concentrations constant. Control samples do notcontain the test compound. Ten microliters of the mixture is then addedto the bottom of a well of a Corning Costar ultra low binding 96-wellplate (Corning Costar, Cambridge Mass.; cat. No. 2500). Ninetymicroliters of water is added to the well, the plate is shaken on arotary shaken at a constant speed at room temperature for 30 seconds, anadditional 100 μl of 2×PTL buffer (20 mM NaH₂PO₄, 300 mM NaCl, pH 7.4)is added to the well, the plate is reshaken for 30 seconds and abaseline (t=0) turbidity reading is taken by measuring the apparentabsorbance at 405 nm using a Bio-Rad Model 450 Microplate Reader. Theplate is then returned to the shaker and shaken continuously for 5hours. Turbidity readings are taken at 15 minute intervals.

[0305] β-amyloid aggregation in the absence of any modulators results inenhanced turbidity of the natural β-AP solution (i.e., an increase inthe apparent absorbance at 405 nm over time). Accordingly, a solutionincluding an effective inhibitory modulator compound exhibits reducedturbidity as compared to the control sample without the modulatorcompound (i.e., less apparent absorbance at 405 nm over time as comparedto the control sample).

[0306] B. Seeded Extension Assay

[0307] The seeded extension assay can be employed to measure the rate ofAβ fiber formed in a solution of Aβ monomer following addition ofpolymeric Aβ fiber “seed”. The ability of test compounds to preventfurther deposition of monomeric AP to previously deposited amyloid isdetermined using a direct indicator of β-sheet formation usingfluorescence. In contrast with the nucleation assay, the addition ofseed provides immediate nucleation and continued growth of preformedfibrils without the need for continuous mixing, and thus results in theabsence of a lag time before polymerization starts. Since this assayuses static polymerization conditions, the activity of positivecompounds in the nucleation assay can be confirmed in this second assayunder different conditions and with an additional probe of amyloidstructure.

[0308] In the seeded extension assay, monomeric Aβ₁₋₄₀ is incubated inthe presence of a “seed” nucleus (approximately ten mole percent of Aβthat has been previously allowed to polymerize under controlled staticconditions). Samples of the solution are then diluted in thioflavin T(Th-T). The polymer-specific association of Th-T with Aβ produces afluorescent complex that allows the measurement of the extent of fibrilformation (Levine, H. (1993) Protein Science 2:404-410). In particular,association of Th-T with aggregated β-AP, but not monomeric or looselyassociated β-AP, gives rise to a new excitation (ex) maximum at 450 nmand an enhanced emission (em) at 482 nm, compared to the 385 nm (ex) and445 nm (em) for the free dye. Small aliquots of the polymerizationmixture contain sufficient fibril to be mixed with Th-T to allow themonitoring of the reaction mixture by repeated sampling. A linear growthcurve is observed in the presence of excess monomer. The formation ofthioflavin T responsive β-sheet fibrils parallels the increase inturbidity observed using the nucleation assay.

[0309] A solution of Aβ monomer for use in the seeded extension assay isprepared by dissolving an appropriate quantity of Aβ₁₋₄₀ peptide in{fraction (1/25)} volume of dimethysulfoxide (DMSO), followed by waterto ½ volume and ½ volume 2×PBS (10×PBS: NaCl 137 mM, KCl 2.7 mMNa₂HPO₄.7H₂O 4.3 mM, KH₂PO₄ 1.4 mM pH 7.2) to a final concentration of200 μM. To prepare the stock seed, 1 ml of the Aβ monomer preparation,is incubated for approximately 8 days at 37° C. and sheared sequentiallythrough an 18, 23, 26 and 30 gauge needle 25, 25, 50, and 100 timesrespectively. 2 μl samples of the sheared material is taken forfluorescence measurements after every 50 passes through the 30 gaugeneedle until the fluorescence units (FU) plateau (approx. 100-150×).Test compounds are prepared by dissolving an appropriate amount of testcompound in 1×PBS to a final concentration of 1 mM (10× stock). Ifinsoluble, the compound is dissolved in {fraction (1/10)} volume of DMSOand diluted in 1×PBS to 1 mM. A further {fraction (1/10)} dilution isalso prepared to test each candidate at both 100 μM and 10 μM.

[0310] To perform the seeded extension assay, each sample is set up with50 μl of 200 μM monomer, 125 FU sheared seed (a variable quantitydependent on the batch of seed, routinely 3-6 μl) and 10 μl of 10×modulator solution. The sample volume is then adjusted to a final volumeof 100 μl with 1×PBS. Two concentrations of each modulator typically aretested: 100 μM and 10 μM, equivalent to a 1:1 and a 1:10 molar ratio ofmonomer to modulator. The controls include an unseeded reaction toconfirm that the fresh monomer contains no seed, and a seeded reactionin the absence of any modulators, as a reference to compare againstcandidate modulators. The assay is incubated at 37° C. for 6 h, taking 2μl samples hourly for fluorescence measurements. To measurefluorescence, a 2 μl sample of Aβ is added to 400 μl of Thioflavin-Tsolution (50 mM Potassium Phosphate 10 mM Thioflavin-T pH 7.5). Thesamples are vortexed and the fluorescence is read in a 0.5 ml microquartz cuvette at EX 450 nm and EM 482 nm (Hitachi 4500 Fluorimeter).

[0311] β-amyloid aggregation results in enhanced emission ofThioflavin-T. Accordingly, samples including an effective inhibitorymodulator compound exhibit reduced emission as compared to controlsamples without the modulator compound.

EXAMPLE 7 Effect of Different Amino Acid Subregions of Aβ Peptide on theInhibitory Activity of β-Amyloid Modulator Compounds

[0312] To determine the effect of various subregions of Aβ₁₋₄₀ on theinhibitory activity of a a β-amyloid modulator, overlapping Aβ peptide15mers were constructed. For each 15mer, four different amino-terminalmodifiers were tested: a cholyl group, an iminobiotinyl group, anN-acetyl neuraminyl group (NANA) and a 5-(and 6-)-carboxyfluoresceinylgroup (FICO). The modulators were evaluated in the nucleation and seededextension assays described in Example 6.

[0313] The results of the nucleation assays are summarized below inTable II. The concentration of Aβ₁₋₄₀ used in the assays was 50 μM. The“mole %” value listed in Table II refers to the % concentration of thetest compound relative to Aβ₁₋₄₀. Accordingly, 100% indicates thatAβ₁₋₄₀ and the test compound were equimolar. Mole % values less than100% indicate that Aβ₁₋₄₀ was in molar excess relative to the testcompound (e.g., 10% indicates that Aβ₁₋₄₀ was in 10-fold molar excessrelative to the test compound). The results of the nucleation assays foreach test compound are presented in Table II in two ways. The “foldincrease in lag time”, which is a measure of the ability of the compoundto delay the onset of aggregation, refers to the ratio of the observedlag time in the presence of the test compound to the observed lag timein the control without the test compound. Accordingly a fold increase inlag time of 1.0 indicates no change in lag time, whereas numbers >1.0indicate an increase in lag time. The “% inhibition of plateau”, whichis a measure of the ability of the compound to decrease the total amountof aggregation, refers to the reduction of the final turbidity in thepresence of the test compound expressed as a percent of the controlwithout the test compound. Accordingly, an inhibitor that abolishesaggregation during the course of the assay will have a % inhibition of100. N-terminally modified AP subregions which exhibited inhibitoryactivity are indicated in bold in Table II. TABLE II Fold % ReferenceN-terminal Mole Increase in Inhibition # Modification Aβ Peptide % LagTime of Plateau PPI-174 cholyl Aβ₁₋₁₅ 100 >4.5 100 PPI-264 cholyl Aβ₆₋₂₀100 >4.5 100 PPI-269 cholyl Aβ₁₁₋₂₅ 100 1.5 ˜0 PPI-274 cholyl Aβ₁₆₋₃₀100 >4.5 100 PPI-279 cholyl Aβ₂₁₋₃₅ 100 1.6 51 PPI-284 cholyl Aβ₂₆₋₄₀100 >4.5 87 PPI-173 NANA Aβ₁₋₁₅ 100 ˜1 ˜0 PPI-266 NANA Aβ₆₋₂₀ 100 1.3 64PPI-271 NANA Aβ₁₁₋₂₅ 100 1.3 77 PPI-276 NANA Aβ₁₆₋₃₀ 100 ˜1 ˜0 PPI-281NANA Aβ₂₁₋₃₅ 100 ˜1 53 PPI-286 NANA Aβ₂₆₋₄₀ 100 1.3 ˜0 PPI-172Iminobiotinyl Aβ₁₋₁₅ 100 1.2 ˜0 PPI-267 Iminobiotinyl Aβ₆₋₂₀ 100 1.6 44PPI-272 Iminobiotinyl Aβ₁₁₋₂₅ 100 1.2 40 PPI-277 Iminobiotinyl Aβ₁₆₋₃₀100 1.2 55 PPI-282 Iminobiotinyl Aβ₂₁₋₃₅ 100 ˜1 66 PPI-287 IminobiotinylAβ₂₆₋₄₀ 100 2.3 ˜0 PPI-190 FICO Aβ₁₋₁₅ 100 ˜1 30 PPI-268 FICO Aβ₆₋₂₀ 1001.9 ˜0 PPI-273 FICO Aβ₁₁₋₂₅ 100 1.7 34 PPI-278 FICO Aβ₁₆₋₃₀ 100 1.6 59PPI-283 FICO Aβ₂₁₋₃₅ 100 1.2 25 PPI-288 FICO Aβ₂₆₋₄₀ 100 2 75

[0314] These results indicate that certain subregions of Aβ₁₋₄₀, whenmodified with an appropriate modifying group, are effective atinhibiting the aggregation of Aβ₁₋₄₀. A cholyl group was an effectivemodifying group for several subregions. Cholic acid alone was tested forinhibitory activity but had no effect on Aβ aggregation. The Aβ₆₋₂₀subregion exhibited high levels of inhibitory activity when modifiedwith several different modifying groups (cholyl, NANA, iminobiotinyl),with cholyl-Aβ₆₋₂₀ (PPI-264) being the most active form. Accordingly,this modulator compound was chosen for further analysis, described inExample 8.

EXAMPLE 8 Identification of a Five Amino Acid Subregion of Aβ PeptideSufficient for Inhibitory Activity of a β-Amyloid Modulator Compound

[0315] To further delineate a minimal subregion of cholyl-Aβ₆₋₂₀sufficient for inhibitory activity, a series of amino terminal andcarboxy terminal amino acid deletions of cholyl-Aβ₆₋₂₀ were constructed.The modulators all had the same cholyl amino-terminal modification.Additionally, for the peptide series having carboxy terminal deletions,the carboxy terminus was further modified to an amide. The modulatorswere evaluated as described in Example 7 and the results are summarizedbelow in Table III, wherein the data is presented as described inExample 7. TABLE III Fold % N-Term. Aβ C-Term. Mole Increase inInhibition Ref. # Mod. Peptide Mod. % Lag Time of Plateau PPI-264 cholylAβ₆₋₂₀ — 100 >4.5 100 10 2 43 PPI-341 cholyl Aβ₇₋₂₀ — 100 >4.5 100 33 2˜0 PPI-342 cholyl Aβ₈₋₂₀ — 100 1.5 ˜0 33 2.1 ˜0 PPI-343 cholyl Aβ₉₋₂₀ —33 2.0 ˜0 PPI-344 cholyl Aβ₁₀₋₂₀ — 33 2.1 ˜0 PPI-345 cholyl Aβ₁₁₋₂₀ — 331.5 ˜0 PPI-346 cholyl Aβ₁₂₋₂₀ — 33 2.1 ˜0 PPI-347 cholyl Aβ₁₃₋₂₀ — 332.6 ˜0 PPI-348 cholyl Aβ₁₄₋₂₀ — 33 2.0 49 PPI-349 cholyl Aβ₁₅₋₂₀ — 332.3 50 PPI-350 cholyl Aβ₁₆₋₂₀ — 38 3.4 23 PPI-296 cholyl Aβ₆₋₂₀ amide 331.8 ˜0 PPI-321 cholyl Aβ₆₋₁₉ amide 33 1.4 ˜0 PPI-325 cholyl Aβ₆₋₁₇ amide33 1.8 ˜0 PPI-331 cholyl Aβ₆₋₁₄ amide 33 1.0 29 PPI-339 cholyl Aβ₆₋₁₀amide 33 1.1 13

[0316] These results indicate that activity of the modulator ismaintained when amino acid residue 6 is removed from the amino terminalend of the modulator (i.e., cholyl-Aβ₇₋₂₀ retained activity) butactivity is lost as the peptide is deleted further at the amino-terminalend by removal of amino acid position 7 through to amino acid position12 (i.e., cholyl-Aβ₈₋₂₀ through cholyl-Aβ₁₃₋₂₀ did inhibit the plateaulevel of AP aggregation). However, further deletion of amino acidposition 13 resulted in a compound (i.e., cholyl-Aβ₁₄₋₂₀) in whichinhibitory activity is restored. Furthermore, additional deletion ofamino acid position 14 (i.e., cholyl-Aβ₁₅₋₂₀) or positions 14 and 15(i.e., cholyl-Aβ₁₆₋₂₀) still maintained inhibitory activity. Thus, aminoterminal deletions of Aβ₆₋₂₀ identified Aβ₁₆₋₂₀ as a minimal subregionwhich is sufficient for inhibitory activity when appropriately modified.In contrast, carboxy terminal deletion of amino acid position 20resulted in loss of activity which was not fully restored as the peptidewas deleted further at the carboxy-terminal end. Thus, maintenance ofposition 20 within the modulator may be important for inhibitoryactivity.

EXAMPLE 9 Identification of a Four Amino Acid Subregion of Aβ PeptideSufficient for Inhibitory Activity of a β-Amyloid Modulator Compound

[0317] In this example, the smallest effective modulator identified inthe studies described in Example 8, cholyl-Aβ₁₆₋₂₀ (PPI-350), wasanalyzed further. Additional amino- and carboxy-terminal deletions weremade with cholyl-Aβ₁₆₋₂₀, as well as an amino acid substitution(Val₁₈->Thr), to identify the smallest region sufficient for theinhibitory activity of the modulator. A peptide comprised of fivealanine residues, (Ala)₅, modified at its amino-terminus with cholicacid, was used as a specificity control. The modulators were evaluatedas described in Example 7 and the results are summarized below in TableIV, wherein the data is presented as described in Example 7. TABLE IV C-Fold % N-Term. Aβ Term. Mole Increase in Inhibition Ref. # Mod. PeptideMod. % Lag Time of Plateau PPI-264 cholyl Aβ₆₋₂₀ — 10 2.0 43 PPI-347cholyl Aβ₁₃₋₂₀ — 10 2.2 57 PPI-349 cholyl Aβ₁₅₋₂₀ — 100 >5.0 100 33 2.635 10 2.1 ˜0 PPI-350 cholyl Aβ₁₆₋₂₀ — 100 >5.0 100 10 2.4 40 PPI-368cholyl Aβ₁₇₋₂₁ — 100 >5.0 100 PPI-374 imino- Aβ₁₆₋₂₀ — 100 1.3 86biotinyl PPI-366 cholyl Aβ₁₅₋₁₉ — 100 3.1 ˜0 10 1.6 ˜0 PPI-369 cholylAβ₁₆₋₂₀ — 100 ˜1 ˜0 (Val₁₈−> Thr) PPI-370 cholyl Aβ₁₆₋₂₀ — 100 2.6 73(Phe₁₉−> Ala) PPI-365 cholyl (Ala)₅ — 100 ˜1 ˜0 PPI-319 cholyl Aβ₁₆₋₂₀amide 33 5.6 ˜0 10 2.7 ˜0 PPI-321 cholyl Aβ₁₆₋₁₉ amide 100 1.2 ˜0PPI-377 — Aβ₁₆₋₂₀ — 100 ˜1 ˜0

[0318] As shown in Table IV, cholyl-Aβ₁₆₋₂₀ (PPI-350) and cholyl-Aβ₁₇₋₂₁(PPI-368) both exhibited inhibitory activity, indicating that thefour-amino acid minimal subregion of positions 17-20 is sufficient forinhibitory activity. Loss of position 20 (e.g., in PPI-366 and PPI-321)resulted in loss of inhibitory activity, demonstrating the importance ofposition 20. Moreover, mutation of valine at position 18 to threonine(in PPI-369) also resulted in loss of activity, demonstrating theimportance of position 18. In contrast, mutation of phenylalanine atposition 19 to alanine (cholyl-Aβ₁₆₋₂₀ Phe₁₉->Ala; PPI-370) resulted ina compound which still retained detectable inhibitory activity.Accordingly, the phenylalanine at position 19 is more amenable tosubstitution, preferably with another hydrophobic amino acid residue.Cholyl-penta-alanine (PPI-365) showed no inhibitory activity,demonstrating the specificity of the AP peptide portion of themodulator. Moreover, unmodified Aβ₁₆₋₂₀ (PPI-377) was not. inhibitory,demonstrating the functional importance of the amino-terminal modifyinggroup. The specific functional group influenced the activity of themodulator. For example, iminobiotinyl-Aβ₁₆₋₂₀ (PPI-374) exhibitedinhibitory activity similar to cholyl-Aβ₁₆₋₂₀, whereas an N-acetylneuraminic acid (NANA)-modified Aβ₁₆₋₂₀ was not an effective inhibitorymodulator (not listed in Table IV). A C-terminal amide derivative ofcholyl-Aβ₁₆₋₂₀ (PPI-319) retained high activity in delaying the lag timeof aggregation, indicating that the carboxy-terminus of the modulatorcan be derivatized without loss of inhibitory activity. Although thisamide-derivatized compound did not inhibit the overall plateau level ofaggregation over time, the compound was not tested at concentrationshigher than mole 33%. Higher concentrations of the amide-derivatizedcompound are predicted to inhibit the overall plateau level ofaggregation, similar to cholyl-Aβ₁₆₋₂₀ (PPI-350).

EXAMPLE 10 Effect of β-Amyloid Modulators on the Neurotoxicity ofNatural β-Amyloid Peptide Aggregates

[0319] The neurotoxicity of natural β-amyloid peptide aggregates, ineither the presence or absence of a β-amyloid modulator, is tested in acell-based assay using either a rat or human neuronally-derived cellline (PC-12 cells or NT-2 cells, respectively) and the viabilityindicator 3,(4,4-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide(MTT). (See e.g., Shearman, M. S. et al (1994) Proc. Natl. Acad. Sci.USA 91:1470-1474; Hansen, M. B. et al. (1989) J. Immun. Methods119:203-210 for a description of similar cell-based viability assays).PC-12 is a rat adrenal pheochromocytoma cell line and is available fromthe American Type Culture Collection, Rockville, Md. (ATCC CRL 1721).MTT (commercially available from Sigma Chemical Co.) is a chromogenicsubstrate that is converted from yellow to blue in viable cells, whichcan be detected spectrophotometrically.

[0320] To test the neurotoxicity of natural β-amyloid peptides, stocksolutions of fresh Aβ monomers and aged Aβ aggregates were firstprepared. Aβ₁₋₄₀ in 100% DMSO was prepared from lyophilized powder andimmediately diluted in one half the final volume in H₂O and then onehalf the final volume in 2×PBS so that a final concentration of 200 μMpeptide, 4% DMSO is achieved. Peptide prepared in this way and testedimmediately on cells is referred to as “fresh” Aβ monomer. To prepare“aged” Aβ aggregates, peptide solution was placed in a 1.5 ml Eppendorftube and incubated at 37° C. for eight days to allow fibrils to form.Such “aged” Aβ peptide can be tested directly on cells or frozen at −80°C. The neurotoxicity of fresh monomers and aged aggregates were testedusing PC12 and NT2 cells. PC12 cells were routinely cultured inDulbeco's modified Eagle's medium (DMEM) containing 10% horse serum, 5%fetal calf serum, 4 mM glutamine, and 1% gentamycin. NT2 cells wereroutinely cultured in OPTI-MEM medium (GIBCO BRL CAT. #31985)supplemented with 10% fetal calf serum, 2 mM glutamine and 1%gentamycin. Cells were plated at 10-15,000 cells per well in 90 μl offresh medium in a 96-well tissue culture plate 3-4 hours prior totreatment. The fresh or aged Aβ peptide solutions (10 μL) were thendiluted 1:10 directly into tissue culture medium so that the finalconcentration was in the range of 1-10 μM peptide. Cells are incubatedin the presence of peptide without a change in media for 48 hours at 37°C. For the final three hours of exposure of the cells to the β-APpreparation, MTT was added to the media to a final concentration of 1mg/ml and incubation was continued at 37° C. Following the two hourincubation with MTT, the media was removed and the cells were lysed in100 μL isopropanol/0.4N HCl with agitation. An equal volume of PBS wasadded to each well and the plates were agitated for an additional 10minutes. Absorbance of each well at 570 nm was measured using amicrotiter plate reader to quantitate viable cells.

[0321] The neurotoxicity of aged (5 day or 8 day) Aβ₁₋₄₀ aggregatesalone, but not fresh Aβ₁₋₄₀ monomers alone, was confirmed in anexperiment the results of which are shown in FIG. 3, which demonstratesthat incubating the neuronal cells with increasing amounts of freshAβ₁₋₄₀ monomers was not significantly toxic to the cells whereasincubating the cells with increasing amounts of 5 day or 8 day Aβ₁₋₄₀aggregates led to increasing amount of neurotoxicity. The EC50 fortoxicity of aged Aβ₁₋₄₀ aggregates was 1-2 μM for both the PC12 cellsand the NT2 cells.

[0322] To determine the effect of a β-amyloid modulator compound on theneurotoxicity of Aβ₁₋₄₀ aggregates, a modulator compound, cholyl-Aβ₆₋₂₀(PPI-264), was preincubated with Aβ₁₋₄₀ monomers under standardnucleation assay conditions as described in Example 6 and at particulartime intervals post-incubation, aliquots of the β-AP/modulator solutionwere removed and 1) the turbidity of the solution was assessed as ameasure of aggregation and 2) the solution was applied to culturedneuronal cells for 48 hours at which time cell viability was assessedusing MTT to determine the neurotoxicity of the solution. The results ofthe turbidity analysis are shown in FIG. 4, panels A, B and C. In panelA, Aβ₁₋₄₀ and cholyl-Aβ₆₋₂₀ were both present at 64 μM. In panel B,Aβ₁₋₄₀ was present at 30 μM and cholyl-Aβ₆₋₂₀ was present at 64 μM. Inpanel C, Aβ₁₋₄₀ was present at 10 μM and cholyl-Aβ₆₋₂₀ was present at 64μM. These data show that an equimolar amount of cholyl-Aβ₆₋₂₀ iseffective at inhibiting aggregation of Aβ₁₋₄₀ (see FIG. 4, panel A) andthat as the concentration of Aβ₁₋₄₀ is reduced, the amount of detectableaggregation of the Aβ₁₋₄₀ monomer is correspondingly reduced (compareFIG. 4, panels B and C with panel A). The corresponding results of theneurotoxicity analysis are shown in FIG. 4, panels D, E, and F. Theseresults demonstrate that the β-amyloid modulator compound not onlyinhibits aggregation of Aβ₁₋₄₀ monomers but also inhibits theneurotoxicity of the Aβ₁₋₄₀ solution, illustrated by the reduced percenttoxicity of the cells when incubated with the Aβ₁₋₄₀/modulator solutionas compared to Aβ₁₋₄₀ alone (see e.g., FIG. 4, panel D). Moreover, evenwhen Aβ₁₋₄₀ aggregation was not detectable as measured by lightscattering, the modulator compound inhibited the neurotoxicity of theAβ₁₋₄₀ solution (see. FIG. 4, panels E and F). Thus, the formation ofneurotoxic Aβ₁₋₄₀ aggregates precedes the formation of insolubleaggregates detectable by light scattering and the modulator compound iseffective at inhibiting the inhibiting the formation and/or activity ofthese neurotoxic aggregates. Similar results were seen with othermodulator compounds, such as iminobiotinyl-Aβ₆₋₂₀ (PPI-267),cholyl-Aβ₁₆₋₂₀ (PPI-350) and cholyl-Aβ₁₆₋₂₀-amide (PPI-319).

[0323] Additionally, the β-amyloid modulator compounds have beendemonstrated to reduce the neurotoxicity of preformed Aβ₁₋₄₀ aggregates.In these experiments, Aβ₁₋₄₀ aggregates were preformed by incubation ofthe monomers in the absence of any modulators. The modulator compoundwas then incubated with the preformed Aβ₁₋₄₀ aggregates for 24 hours at37° C., after which time the β-AP/modulator solution was collected andits neurotoxicity evaluated as described above. Incubation of preformedAβ₁₋₄₀ aggregates with the modulator compound prior to applying thesolution to neuronal cells resulted in a decrease in the neurotoxicityof the Aβ₁₋₄₀ solution. These results suggest that the modulator caneither bind to Aβ fibrils or soluble aggregate and modulate theirinherent neurotoxicity or that the modulator can perturb the equilibriumbetween monomeric and aggregated forms of Aβ₁₋₄₀ in favor of thenon-neurotoxic form.

EXAMPLE 11 Characterization of Additional β-Amyloid Modulator Compounds

[0324] In this example, additional modulator compounds designed basedupon amino acids 17-20 of Aβ, LVFF (identified in Example 9), wereprepared and analyzed to further delineate the structural featuresnecessary for inhibition of β-amyloid aggregation. Types of compoundsanalyzed included ones having only three amino acid residues of an Aβaggregation core domain, compounds in which the amino acid residues ofan Aβ aggregation core domain were rearranged or in which amino acidsubstitutions had been made, compounds modified with a carboxy-terminalmodifying group and compounds in which the modifying group had beenderivatized. Abbreviations used in this example are: h- (free aminoterminus), -oh (free carboxylic acid terminus), -nh₂ (amide terminus),CA (cholyl, the acyl portion of cholic acid), NANA (N-acetylneuraminyl), IB (iminobiotinyl), βA (β-alanyl), DA (D-alanyl), Adp(aminoethyldibenzofuranylpropanoic acid), Aic(3-(O-aminoethyl-iso)-cholyl, a derivative of cholic acid), IY(iodotyrosyl), o-methyl (carboxy-terminal methyl ester), N-me (N-methylpeptide bond), DeoxyCA (deoxycholyl) and LithoCA (lithocholyl).

[0325] Modulator compounds having an Aic modifying group at either theamino- or carboxy-terminus (e.g., PPI-408 and PPI-418) were synthesizedusing known methods (see e.g., Wess, G. et al. (1993) TetrahedronLetters, 34:817-822; Wess, G. et al. (1992) Tetrahedron Letters33:195-198). Briefly, 3-iso-O-(2-aminoethyl)-cholic acid(3β-(2-aminoethoxy)-7α,12α-dihydroxy-5β-cholanoic acid) was converted tothe FMOC-protected derivative using FMOC-OSu (the hydroxysuccinimideester of the FMOC group, which is commercially available) to obtain areagent that was used to introduce the cholic acid derivative into thecompound. For N-terminal introduction of the cholic acid moiety, theFMOC-protected reagent was coupled to the N-terminal amino acid of asolid-phase peptide in the standard manner, followed by standardFMOC-deprotection conditions and subsequent cleavage from the resin,followed by HPLC purification. For C-terminal introduction of the cholicacid moiety, the FMOC-protected reagent was attached to 2-chlorotritylchloride resin in the standard manner. This amino acyl derivatized resinwas then used in the standard manner to synthesize the complete modifiedpeptide.

[0326] The modulators were evaluated in the nucleation and seededextension assays described in Example 6 and the results are summarizedbelow in Table V. The change in lag time (ΔLag) is presented as theratio of the lag time observed in the presence of the test compound tothe lag time of the control. Data are reported for assays in thepresence of 100 mole % inhibitor relative to the concentration ofAβ₁₋₄₀, except for PPI-315, PPI-348, PPI-380, PPI-407 and PPI-418, forwhich the data is reported in the presence of 33 mole % inhibitor.Inhibition (% I_(nucl'n)) is listed as the percent reduction in themaximum observed turbidity in the control at the end of the assay timeperiod. Inhibition in the extension assay (% I_(ext'n)) is listed as thepercent reduction of thioflavin-T fluorescence of β-structure in thepresence of 25 mole % inhibitor. Compounds with a % I_(nucl'n) of atleast 30% are highlighted in bold. TABLE V N-Term. C-Term. Ref. # Mod.Peptide Mod. ΔLag % I_(nucl'n) % I_(ext'n) PPI-293 CA — -oh 1.0 0 ND*PPI-315 CA HQKLVFF -nh₂ 1.1  5** ND PPI-316 NANA HQKLVFF -nh₂ 1.5 −15 NDPPI-319 CA KLVFF -nh₂ 5.4 70 52 PPI-339 CA HDSGY -nh₂ 1.1 −18 ND PPI-348CA HQKLVFF -oh 2.0  70** ND PPI-349 CA QKLVFF -oh >5 100 56 PPI-350 CAKLVFF -oh 1.8 72 11 PPI-365 CA AAAAA -oh 0.8 −7 0 PPI-366 CA QKLVF -oh3.1 −23 ND PPI-368 CA LVFFA -oh >5 100 91 PPI-369 CA KLTFF oh 1.1 −16 44PPI-370 CA KLVAF -oh 2.6 73 31 PPI-371 CA KLVFF(βA) -oh 2.5 76 80PPI-372 CA FKFVL -oh 0.8 45 37 PPI-373 NANA KLVFF -oh 0.9 16 8 PPI-374IB KLVFF -oh 1.3 86 0 PPI-375 CA KTVFF -oh 1.2 18 21 PPI-377 h- KLVFF-oh 1.1 0 8 PPI-379 CA LVFFAE -oh 1.4 55 16 PPI-380 CA LVFF -oh 1.8 72** 51 PPI-381 CA LVFF(DA) -oh 2.3 56 11 PPI-382 CA LVFFA -nh₂ 1.0−200 91 PPI-383 h-DDIIL- VFF -oh 0.4 14 0 (Adp) PPI-386 h- LVFFA -oh 1.015 11 PPI-387 h- KLVFF -nh₂ 1.3 −9 39 PPI-388 CA AVFFA -oh 1.4 68 44PPI-389 CA LAFFA -oh 1.5 47 66 PPI-390 CA LVAFA -oh 2.7 25 0 PPI-392 CAVFFA -oh 2.0 76 10 PPI-393 CA LVF -oh 1.3 1 0 PPI-394 CA VFF -oh 1.8 550 PPI-395 CA FFA -oh 1.0 51 6 PPI-396 CA LV(IY)FA -oh >5 100 71 PPI-401CA LVFFA -o-methyl ND ND 0 PPI-405 h- LVFFA -nh₂ 1.3 11 70 PPI-407 CALVFFK -oh >5 100** 85 PPI-408 h- LVFFA (Aic)-oh 3.5 46 3 PPI-418 h-(Aic)LVFFA -oh >5 100** 87 PPI-426 CA FFVLA -oh >5 100 89 PPI-391 CA LVFAA-oh 1.6 40 ND PPI-397 CA LVF(IY)A -oh >5 95 ND PPI-400 CA AVAFA -oh 1.0−15 ND PPI-403 *** HQKLVFF -oh 1.4 −75 0 PPI-404 **** LKLVFF -oh 1.8 −297 PPI-424 DeoxyCA LVFFA -oh 3.0 −114 82 PPI-425 LithoCA LVFFA -oh 2.8−229 0 PPI-428 CA FF -oh 1.7 −78 15 PPI-429 CA FFV -oh 2.2 −33 7 PPI-430CA FFVL -oh 4.1 33 75 PPI-433 CA LVFFA -oh 2.8 27 ND (all D amino acids)PPI-435 t-Boc LVFFA -oh 3.0 −5 ND PPI-438 CA GFF -oh 1.0 0 ND

[0327] Certain compounds shown in Table V (PPI-319, PPI-349, PPI-350,PPI-368 and PPI-426) also were tested in neurotoxicity assays such asthose described in Example 10. For each compound, the delay of theappearance of neurotoxicity relative to control coincided with the delayin the time at which polymerization of Aβ began in the nucleationassays. This correlation between the prevention of formation ofneurotoxic Aβ species and the prevention of polymerization of Aβ wasconsistently observed for all compounds tested.

[0328] The results shown in Table V demonstrate that at an effectivemodulator compound can comprise as few as three Aβ amino acids residues(see PPI-394, comprising the amino acid sequence VFF, which correspondsto Aβ₁₈₋₂₀, and PPI-395, comprising the amino acid sequence FFA, whichcorresponds to Aβ₁₉₋₂₁). The results also demonstrate that a modulatorcompound having a modulating group at its carboxy-terminus is effectiveat inhibiting AP aggregation (see PPI-408, modified at its C-terminuswith Aic). Still further, the results demonstrate that the cholyl group,as a modulating group, can be manipulated while maintaining theinhibitory activity of the compounds (see PPI-408 and PPI-418, both ofwhich comprise the cholyl derivative Aic). The free amino group of theAic derivative of cholic acid represents a position at which a chelationgroup for ^(99m)Tc can be introduced, e.g., to create a diagnosticagent. Additionally, the ability to substitute iodotyrosyl forphenylalanine at position 19 or 20 of the Aβ sequence (see PPI-396 andPPI-397) while maintaining the ability of the compound to inhibit Aβaggregation indicates that the compound could be labeled withradioactive iodine, e.g., to create a diagnostic agent, without loss ofthe inhibitory activity of the compound.

[0329] Finally, compounds with inhibitory activity were created using Aβderived amino acids but wherein the amino acid sequence was rearrangedor had a substitution with a non-Aβ-derived amino acid. Examples of suchcompounds include PPI-426, in which the sequence of Aβ₁₇₋₂₁ (LVFFA) hasbeen rearranged (FFVLA), PPI-372, in which the sequence of Aβ₁₆₋₂₀(KLVFF) has been rearranged (FKFVL), and PPI-388, -389 and -390, inwhich the sequence of Aβ₁₇₋₂₁ (LVFFA) has been substituted at position17, 18 or 19, respectively, with an alanine residue (AVFFA for PPI-388,LAFFA for PPI-389 and LVAFA for PPI-390). The inhibitory activity ofthese compounds indicate that the presence in the compound of an aminoacid sequence directly corresponding to a portion of Aβ is not essentialfor inhibitory activity, but rather suggests that maintenance of thehydrophobic nature of this core region, by inclusion of amino acidresidues such as phenylalanine, valine, leucine, regardless of theirprecise order, can be sufficient for inhibition of AP aggregation.

EXAMPLE 12 Characterization of β-Amyloid Modulator Compounds Comprisingan Unmodified β-Amyloid Peptide

[0330] To examine the ability of unmodified Aβ peptides to modulateaggregation of natural β-AP, a series of Aβ peptides having amino-and/or carboxy terminal deletions as compared to Aβ₁₋₄₀, or havinginternal amino acids deleted (i.e., noncontiguous peptides), wereprepared. One peptide (PPI-220) had additional, non-Aβ-derived aminoacid residues at its amino-terminus. These peptides all had a free aminogroup at the amino-terminus and a free carboxylic acid at thecarboxy-terminus. These unmodified peptides were evaluated in assays asdescribed in Example 7. The results are summarized below in Table VI,wherein the data is presented as described in Example 7. Compoundsexhibiting at least a 1.5 fold increase in lag time are highlighted inbold. TABLE VI Fold Reference Mole Increase in % Inhibition # Aβ Peptide% Lag Time of Plateau PPI-226 Aβ₆₋₂₀ 100 1.66 76 PPI-227 Aβ₁₁₋₂₅ 100 ˜147 PPI-228 Aβ₁₆₋₃₀ 100 >4.5 100 PPI-229 Aβ₂₁₋₃₅ 100 ˜1 ˜0 PPI-230Aβ₂₆₋₄₀ 100 0.8 ˜0 PPI-231 Aβ₁₋₁₅ 100 ˜1 18 PPI-247Aβ_(1-30, 36-40)(Δ31-35) 100 ˜1 ˜0 PPI-248 Aβ_(1-25, 31-40)(Δ26-30) 1001.58 ˜0 PPI-249 Aβ_(1-20, 26-40)(Δ21-25) 100 2.37 ˜0 PPI-250Aβ_(1-15, 21-40)(Δ16-20) 100 1.55 ˜0 PPI-251 Aβ_(1-10, 16-40)(Δ11-15)100 ˜1.2 ˜0 PPI-252 Aβ_(1-5, 11-40)(Δ6-10) 100 1.9 33 PPI-253 Aβ₆₋₄₀ 1001.9 ˜0 PPI-220 EEVVHHHHQQ-Aβ₁₆₋₄₀ 100 >4 100

[0331] The results shown in Table VI demonstrate that limited portionsof the Aβ sequence can have a significant inhibitory effect on naturalβ-AP aggregation even when the peptide is not modified by a modifyinggroup. Preferred unmodified peptides are Aβ₆₋₂₀ (PPI-226), Aβ₁₆₋₃₀(PPI-228), Aβ_(1-20, 26-40) (PPI-249) and EEVVHHHHQQ-Aβ₁₆₋₄₀ (PPI-220),the amino acid sequences of which are shown in SEQ ID NOs: 4, 14, 15,and 16, respectively.

[0332] Forming part of this disclosure is the appended Sequence Listing,the contents of which are summarize in the Table below. SEQ ID NO: AminoAcids Peptide Sequence 1 43 amino acids Aβ₁₋₄₃ 2 103 amino acids APPC-terminus 3 43 amino acids Aβ₁₋₄₃(19, 20 mutated) 4 HDSGYEVHHQKLVFFAβ₆₋₂₀ 5 HQKLVFFA Aβ₁₄₋₂₁ 6 HQKLVFF Aβ₁₄₋₂₀ 7 QKLVFFA Aβ₁₅₋₂₁ 8 QKLVFFAβ₁₅₋₂₀ 9 KLVFFA Aβ₁₆₋₂₁ 10 KLVFF Aβ₁₆₋₂₀ 11 LVFFA Aβ₁₇₋₂₁ 12 LVFFAβ₁₇₋₂₀ 13 LAFFA Aβ₁₇₋₂₁(V₁₈→A) 14 KLVFFAEDVGSNKGA Aβ₁₆₋₃₀ 15 35 aminoacids Aβ_(1-20, 26-40) 16 35 amino acids EEVVHHHHQQ-βAP₁₆₋₄₀ 17 AGAAAAGAPrP peptide 18 AILSS amylin peptide 19 VFF Aβ₁₈₋₂₀ 20 FFA Aβ₁₉₋₂₁ 21FFVLA Aβ₁₇₋₂₁(scrambled) 22 LVFFK Aβ₁₇₋₂₁(A₂₁→K) 23 LV(IY)FAAβ₁₇₋₂₁(F₁₉→IY) 24 VFFA Aβ₁₈₋₂₁ 25 AVFFA Aβ₁₇₋₂₁(L₁₇→A) 26 LVF(IY)AAβ₁₇₋₂₁(F₂₀→IY) 27 LVFFAE Aβ₁₇₋₂₂ 28 FFVL Aβ₁₇₋₂₀(scrambled) 29 FKFVLAβ₁₆₋₂₀(scrambled) 30 KLVAF Aβ₁₆₋₂₀(F₁₉→A) 31 KLVFF(βA) Aβ₁₆₋₂₁(A₂₁→βA)32 LVFF(DA) Aβ₁₇₋₂₁(A₂₁→DA)

[0333] Equivalents

[0334] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

1 45 43 amino acids amino acid linear peptide internal 1 Asp Ala Glu PheArg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 Leu Val PhePhe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile 20 25 30 Gly Leu MetVal Gly Gly Val Val Ile Ala Thr 35 40 103 amino acids amino acid linearpeptide internal 2 Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser GlyTyr Glu Val 1 5 10 15 His His Gln Lys Leu Val Phe Phe Ala Glu Asp ValGly Ser Asn Lys 20 25 30 Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val ValIle Ala Thr Val 35 40 45 Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys GlnTyr Thr Ser Ile 50 55 60 His His Gly Val Val Glu Val Asp Ala Ala Val ThrPro Glu Glu Arg 65 70 75 80 His Leu Ser Lys Met Gln Gln Asn Gly Tyr GluAsn Pro Thr Tyr Lys 85 90 95 Phe Phe Glu Gln Met Gln Asn 100 43 aminoacids amino acid linear peptide internal Modified site 19 /note= Xaa isa hydrophobic amino acid Modified site 20 /note= Xaa is a hydrophobicamino acid 3 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His GlnLys 1 5 10 15 Leu Val Xaa Xaa Ala Glu Asp Val Gly Ser Asn Lys Gly AlaIle Ile 20 25 30 Gly Leu Met Val Gly Gly Val Val Ile Ala Thr 35 40 15amino acids amino acid linear peptide 4 His Asp Ser Gly Tyr Glu Val HisHis Gln Lys Leu Val Phe Phe 5 10 15 8 amino acids amino acid linearpeptide 5 His Gln Lys Leu Val Phe Phe Ala 5 7 amino acids amino acidlinear peptide 6 His Gln Lys Leu Val Phe Phe 5 7 amino acids amino acidlinear peptide 7 Gln Lys Leu Val Phe Phe Ala 5 6 amino acids amino acidlinear peptide 8 Gln Lys Leu Val Phe Phe 5 6 amino acids amino acidlinear peptide 9 Lys Leu Val Phe Phe Ala 5 5 amino acids amino acidlinear peptide 10 Lys Leu Val Phe Phe 5 5 amino acids amino acid linearpeptide 11 Leu Val Phe Phe Ala 5 4 amino acids amino acid linear peptide12 Leu Val Phe Phe 5 amino acids amino acid linear peptide internal 13Leu Ala Phe Phe Ala 1 5 15 amino acids amino acid linear peptideinternal 14 Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala1 5 10 15 35 amino acids amino acid linear peptide internal 15 Asp AlaGlu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys 1 5 10 15 LeuVal Phe Phe Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly 20 25 30 GlyVal Val 35 35 amino acids amino acid linear peptide internal 16 Glu GluVal Val His His His His Gln Gln Lys Leu Val Phe Phe Ala 1 5 10 15 GluAsp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly 20 25 30 GlyVal Val 35 8 amino acids amino acid linear peptide internal 17 Ala GlyAla Ala Ala Ala Gly Ala 1 5 5 amino acids amino acid linear peptideinternal 18 Ala Ile Leu Ser Ser 1 5 3 amino acids amino acid linearpeptide 19 Val Phe Phe 1 3 amino acids amino acid linear peptide 20 PhePhe Ala 1 5 amino acids amino acid linear peptide 21 Phe Phe Val Leu Ala1 5 5 amino acids amino acid linear peptide 22 Leu Val Phe Phe Lys 1 5 5amino acids amino acid linear peptide Modified site 3 /note= Xaa isiodotyrosyl 23 Leu Val Xaa Phe Ala 1 5 4 amino acids amino acid linearpeptide 24 Val Phe Phe Ala 1 5 amino acids amino acid linear peptide 25Ala Val Phe Phe Ala 1 5 5 amino acids amino acid linear peptide Modifiedsite 4 /note= Xaa is iodotyrosyl 26 Leu Val Phe Xaa Ala 1 6 amino acidsamino acid linear peptide 27 Leu Val Phe Phe Ala Glu 1 5 4 amino acidsamino acid linear peptide 28 Phe Phe Val Leu 1 5 5 amino acids aminoacid linear peptide 29 Phe Lys Phe Val Leu 1 5 5 amino acids amino acidlinear peptide 30 Lys Leu Val Ala Phe 1 5 6 amino acids amino acidlinear peptide Modified site 6 /note= Xaa is beta-alanyl 31 Lys Leu ValPhe Phe Xaa 1 5 amino acids amino acid linear peptide Modified site 5/note= Xaa is D-alanyl 32 Leu Val Phe Phe Xaa 1 5 amino acids amino acidlinear peptide internal 33 Leu Val Ala Phe Ala 1 5 5 amino acids aminoacid linear peptide internal Modified-site 5 /note=aminoethyldibenzofuranyl- proprionic acid modification 34 Asp Asp IleIle Leu 1 5 5 amino acids amino acid linear peptide internal 35 Ala AlaAla Ala Ala 1 5 5 amino acids amino acid linear peptide internal 36 HisAsp Ser Gly Tyr 1 5 5 amino acids amino acid linear peptide internal 37Gln Lys Leu Val Phe 1 5 5 amino acids amino acid linear peptide internal38 Lys Leu Thr Phe Phe 1 5 5 amino acids amino acid linear peptideinternal 39 Lys Thr Val Phe Phe 1 5 5 amino acids amino acid linearpeptide internal 40 Phe Phe Val Leu Ala 1 5 5 amino acids amino acidlinear peptide internal 41 Leu Val Phe Ala Ala 1 5 5 amino acids aminoacid linear peptide internal 42 Ala Val Ala Phe Ala 1 5 6 amino acidsamino acid linear peptide internal 43 Leu Lys Leu Val Phe Phe 1 5 9amino acids amino acid linear peptide internal Modified site 6 /note=Xaa is N-methyl-Val Modified site 9 /note= aminoethyldibenzofuranyl-proprionic acid modification 44 Asp Asp Ile Ile Ile Xaa Asp Leu Leu 1 58 amino acids amino acid linear peptide internal Modified site 5 /note=Xaa is N-Methyl-Leu Modified site 8 /note= aminoethyldibenzofuranyl-proprionic acid modification 45 Asp Asp Ile Ile Xaa Val Glu His 1 5

1. An amyloid modulator compound consisting of the structure:

wherein Xaa is an amyloidogenic protein, or peptide fragment thereof, ofat least 4 amino acid residues in length and a is a modifying groupcomprising a cyclic, heterocyclic or polycyclic group, covalentlyattached to the α-amino group at the amino-terminus of the amyloidogenicprotein, or peptide fragment thereof, such that the compound modulatesthe aggregation of natural amyloid proteins or peptides when contactedwith the natural amyloidogenic proteins or peptides, wherein Xaa is notcalcitonin:
 2. The compound of claim 1 or 7, which inhibits aggregationof natural amyloidogenic proteins or peptides when contacted with thenatural amyloidogenic proteins or peptides.
 3. The compound of claim 2,which inhibits aggregation of natural amyloidogenic proteins or peptideswhen contacted with a molar excess amount of natural amyloidogenicproteins or peptides.
 4. The compound of claim 1 or 7, which is furthermodified to alter a pharmacokinetic property of the compound.
 5. Thecompound of claim 1 or 7, which is further modified to label thecompound with a detectable substance.
 6. The compound of claim 1 or 7,wherein the amyloidogenic protein, or peptide fragment thereof, isselected from the group consisting of transthyretin (TTR), prion protein(PrP), islet amyloid polypeptide (IAPP), atrial natriuretic factor(ANF), kappa light chain, lambda light chain, amyloid A, procalcitonin,cystatin C, β2 microglobulin, ApoA-I, gelsolin, fibrinogen and lysozyme.7. An amyloid modulator compound having the structure:

wherein Xaa is an amyloidogenic protein, or peptide fragment thereof, ofat least 4 amino acid residues in length and A is a modifying groupcomprising a cyclic, heterocyclic or polycyclic group, covalentlyattached to the carboxy-terminus of the amyloidogenic protein, orpeptide fragment thereof, such that the compound modulates theaggregation of natural amyloid proteins or peptides when contacted withthe natural amyloidogenic proteins or peptides, wherein Xaa is notcalcitonin.
 8. The compound of claim 1 or 7, wherein the modifying groupcontains a cis-decalin group.
 9. The compound of claim 8, wherein themodifying group contains a cholanoyl structure.
 10. The compound ofclaim 9, wherein the modifying group is a cholyl group.
 11. The compoundof claim 1 or 7, wherein the modifying group comprises abiotin-containing group, a diethylene-triaminepentaacetyl group, a(−)-menthoxyacetyl group, a fluorescein-containing group or anN-acetylneuraminyl group.
 12. A pharmaceutical composition comprising atherapeutically effective amount of the compound of claim 1 or 7 and apharmaceutically acceptable carrier.
 13. A method for alteringaggregation of natural amyloid proteins or peptides, comprisingcontacting the natural amyloid proteins or peptides with the compound ofclaim 1 or 7 such that aggregation of the natural amyloid proteins orpeptides is altered.
 14. A method for detecting aggregation of naturalamyloid proteins or peptides, comprising contacting a biological samplewith the compound of claim 5 such that aggregation of the naturalamyloid proteins or peptides in the sample is detected.
 15. The methodof claim 14, wherein the compound is administered to a subject to detectaggregation of the natural amyloid proteins or peptides in the subject.16. The method of claim 15, wherein the compound is labeled withradioactive iodine or technetium.
 17. A method for treating a subjectfor a disorder associated with amyloidosis, comprising: administering tothe subject a therapeutically or prophylactically effective amount ofthe compound of claim 1 such that the subject is treated for a disorderassociated with amyloidosis.
 18. A method for treating a subject for adisorder associated with amyloidosis, comprising: administering to thesubject a therapeutically or prophylactically effective amount of thecompound of claim 7 such that the subject is treated for a disorderassociated with amyloidosis.
 19. The method of claim 18, wherein thedisorder is selected from the group consisting of familial amyloidpolyneuropathy (Portuguese, Japanese and Swedish types), familialamyloid cardiomyopathy (Danish type), isolated cardiac amyloid, systemicsenile amyloidosis, scrapie, bovine spongiform encephalopathy,Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome,adult onset diabetes, insulinoma, isolated atrial amyloidosis,idiopathic (primary) amyloidosis, myeloma ormacroglobulinemia-associated amyloidosis, primary localized cutaneousnodular amyloidosis associated with Sjogren's syndrome, reactive(secondary) amyloidosis, familial Mediterranean Fever and familialamyloid nephropathy with urticaria and deafness (Muckle-Wells syndrome),hereditary cerebral hemorrage with amyloidosis of Icelandic type,amyloidosis associated with long term hemodialysis, hereditarynon-neuropathic systemic amyloidosis (familial amyloid polyneuropathyIII), familial amyloidosis of Finnish type, amyloidosis associated withmedullary carcinoma of the thyroid, fibrinogen-associated hereditaryrenal amyloidosis and lysozyme-associated hereditary systemicamyloidosis.